SOCE in skeletal muscle

4.3.1. Discovery, measurements

The first measurements of SOCE in skeletal muscle came belatedly in 2001 with Kurebayashi’s study (Kurebayashi and Ogawa 2001). Before this, SOCE was largely underestimated likely because SR refilling was believed to occur only with Ca²⁺ recycled after contractions and

pumped back into the SR by SERCA pumps (MacLennan 2000). By combining KCl pushes and the utilization in a second time of a SERCA inhibitor, the cyclopiazonic Acid (CPA), Kurebayashi and co-workers succeeded to fully deplete the stores without damaging the muscle fibers. In such conditions, Mn²⁺ quench experiments allowed them to detect Ca²⁺ entry in muscle fibers together with the restoration of the ability of fibers to contract that was initially lost after SR depletion. This study definitively proved the presence of a SOCE in muscle cells but raised many questions concerning the relevance of such influx in this cell type, its characteristics and its activation under more physiological conditions. To answer some of these questions, Launikonis and coworkers adapted a very sensitive technique, firstly developed in 1995 by Lamb’s lab to study ECC that allows simultaneous recording of external and cytosolic Ca²⁺

variations in a muscle fiber (Lamb, Junankar et al. 1995, Launikonis, Barnes et al. 2003). This technique called skin fibers method is based on the large amount (80%) of plasma membrane that form deep-invaginations, the t-tubules, within muscle fibers (Cully and Launikonis 2013).

To obtain skin fibers, muscle fibers are bathed in a typical external solution containing around 1,5mM Ca²⁺ and a low-affinity Ca²⁺ dye, and are peeled off under a paraffin layer to remove the sarcolemma. During the procedure, t-tubules remained in place and sealed off while external medium containing the Ca²⁺ dye is trapped within the new formed structures (the sealed t-tubules). Medium is then changed for internal medium mimicking sarcoplasmic content with low [Na⁺], high [K⁺], 1mM of Mg²⁺, ATP, creatine phosphate and supplemented with high-affinity Ca²⁺ dye. Then, SOCE is elicited by SR depletion (RyR opening through different procedure such as Mg²⁺ removal), and fibers mounted on a confocal microscope.

Fibers are scanned perpendicularly to the longitudinal section for spatial resolution (x axis) and the y axis can be converted into time as the structure of the muscle fiber is homogenous.

Ca²⁺ variations within the cytoplasm and within the sealed t-tubules (that formed a defined space whose ionic variations can be easily recorded) are registered with high temporal resolution (order of ms) (figure III.17).

However, it is important to note the main caveat of skin fibers : loss of some cytoplasmic factors that would be implicated in Ca²⁺ handling and modified SOCE properties determined with the technique (Launikonis, Murphy et al. 2010).

Figure III.17: Ca²⁺movements in mechanically skinned fibers associated with SOCE.

Adapted from Cully T. R., et al. (2013)

Store-operated Ca²⁺ entry (SOCE) is conducted across the tubular (t-) system membrane. Confocal images of the fluorescence of fluo-5 N trapped in the sealed t-system (a) in the presence of a cytoplasmic solution, (b) 30 s after application of a low Mg²⁺ solution containingcaffeine to induce RyR dependent Ca²⁺release and (c–e) after 2 (c), 7 (d) and 12 min (e) after release. Note that depleting the sarcoplasmic reticulum (SR) of Ca²⁺ with caffeine and low Mg²⁺also resulted in the loss of Ca²⁺from the sealed t-system, indicating an operational SOCE (b), and that Ca²⁺can be reloaded into the Ca²⁺ -depleted t-system (c–e) due to PMCA and NCX activity. Bar, 20 μm.

4.3.2. SOCE features in skeletal muscle

 STIM1 and Orai1 are essential players for SOCE. Are they the only one?

Generation of global (Stiber, Hawkins et al. 2008) and muscle specific KO mice (Li, Finch et al.

2012) for STIM1 and Orai1 (Carrell, Coppola et al. 2016) have demonstrated the requirement of both partners as necessary for full activation of SOCE in muscle fibers. In addition, two research groups have succeeded to record by patch-clamp typical ICRAC current (store dependent, inwardly rectifying, with reverse potential at positive voltages, inhibited by known ICRAC inhibitors like BTP-2 and Gd³⁺) on myotubes derived from WT mice (Stiber, Hawkins et al.

2008, Yarotskyy and Dirksen 2012). In these studies, the current was lost after STIM1 silencing or over-expression of a dominant negative Orai1 mutant. In another related study, the authors showed by Ca²⁺ imaging experiments that STIM1 silencing or over-expression of a dominant negative Orai1 mutant abolished SOCE without altering Excitation-coupled Ca²⁺ entry or ECCE

that is another Ca²⁺ entry pathways in skeletal muscle triggered upon repetitive or sustained depolarizations. Thus, although STIM1 and Orai1 are essential players for SOCE in skeletal muscle, one can note that at least in myoblasts other Orai and STIM isoforms/genes: Orai3, STIM1L, STIM2 play a significant role in Ca²⁺ handling and myogenesis (Darbellay, Arnaudeau et al. 2009, Darbellay, Arnaudeau et al. 2010, Darbellay, Arnaudeau et al. 2011, Antigny, Sabourin et al. 2017). Finally, TRPC channels, at least TRPC1 and TRPC4, are known to participate to SOCE in muscle fibers (Vandebrouck, Martin et al. 2002, Vandebrouck, Sabourin et al. 2007, Sabourin, Lamiche et al. 2009) and are regulated by STIM1 and STIM1L in myoblasts and myotubes (Antigny, Koenig et al. 2013, Antigny, Sabourin et al. 2017). TRPC channels could underlie the linear current recorded by patch-clamp and evoked by Tg application in skeletal mouse myotubes (Stiber, Hawkins et al. 2008).

 SOCE kinetics in skeletal muscle

As mentioned previously, numerous SOCE features have been deciphered thanks to Launikonis’ and co-workers’ studies in skin fibers. More specifically, they were able to point out the fast kinetics of SOCE typical of muscle cells. Indeed, SOCE kinetics appear to be much faster (less than 27ms) in muscle fibers compared to non-excitable cells in which it takes 1 to 2min to develop (Launikonis and Rios 2007, Edwards, Murphy et al. 2010). This feature is in accordance with a pre-localization of STIM1 near Orai1 channels in the terminal cisternae of SR (Stiber, Hawkins et al. 2008). Indeed, any physical uncoupling between t-tubules and SR terminal cisternae either by application of an internal high Ca²⁺ concentration solution to the skin fibers (Launikonis, Barnes et al. 2003) or triad malformation in intact myofibers derived from Mitsugumin 29 or Junctophilin 1 and 2 (all proteins needed for proper triads formation) KO mice hinders SOCE (Pan, Yang et al. 2002, Hirata, Brotto et al. 2006). However, despite a proximal localization of STIM1 and Orai1 in muscle fibers, it seems that they do not interact prior to SR Ca²⁺ depletion as nicely demonstrated in Wei-Lapierre study (Wei-Lapierre, Carrell et al. 2013). Using bimolecular fluorescence complementation (BiFC) approach in which the N-terminal part of YFP was fused to STIM1 and the C-terminal part fused to Orai1, they failed to record any fluorescent signal before SR depletion. On the contrary, upon Ca²⁺ store depletion with Tg, a fluorescent signal was detected due to assembly of the 2 parts of the YFP because of STIM1 and Orai1 interaction. Conversely, other groups failed to record any ICRAC at

the sarcolemma upon SR depletion (Allard, Couchoux et al. 2006, Berbey and Allard 2009) reinforcing the idea of the triad being the major place for SOCE in adult muscle fibers and not the part of sarcolemma that does not forms triads. Finally, STIM1L that have been shown in human primary myotubes to form pre-assembled complex with Orai1 channel at the plasma membrane would play also a major part in the fast SOCE typical of muscle cells because STIM1L (but not STIM1) silencing delays notably SOCE kinetics in myotubes as assessed by Mn²⁺ quench experiments (Darbellay, Arnaudeau et al. 2011).

Thus, SOCE in muscle fibers occurs, at least for the larger part, at the triads following the sequential steps identified with the skin fibers method by Launikonis’ lab (figure III.18).

Figure III.18: Proposed molecular model of SOCE in skeletal muscle.

From Launikonis B. S., et al. (2010)

At high [Ca²⁺] SR, CSQ is in polymers and dimers with significant amounts of Ca²⁺bound. STIM1 and Orai1 monomers in the SR and t-system membranes, respectively, remain separated (a). During the slow, direct release of Ca²⁺ from SR, CSQ polymers break up causing an increase in [Ca²⁺] SR and [Ca²⁺]cyto. The t-system takes up Ca²⁺ (b). SR Ca²⁺ release continues. The weakened buffering power of CSQ in its reduced state sees [Ca²⁺] SR drop, and Ca²⁺ starts to unbind from STIM1 initiating aggregation and movement of these molecules (c). The Ca²⁺ release continues and drops [Ca²⁺] SR passed the sharp threshold for SOCE activation, and Ca²⁺ enters the junctional cleft via the Orai1 complex (e). Following inactivation of the RyR, some Ca²⁺ is resequestered by SR. This is enough Ca²⁺ to deactivate SOCE and shut down the Orai1 Ca²⁺ channel. A net reuptake of Ca²⁺ by the t-system follows (f). Keys to schematic

 SOCE activation and deactivation status

In skeletal muscle fiber, SOCE is activated upon sub maximal SR depletion, with an estimated rate of Ca²⁺ entry at 18.6 μm s−1 (Launikonis and Rios 2007). Then, SOCE activation threshold appears to be dependent on SR Ca²⁺ concentration (Pan, Yang et al. 2002, Edwards, Murphy et al. 2010) but also on RyR1 activity and temperature as ICRAC activation rate increases by 4 times at 37°C compared to RT (20°C) in myotubes (Yarotskyy and Dirksen 2012). Regarding termination of SOCE in muscle cells, no CDI have been reported in physiological Ca²⁺

concentration contrary to what is observed in non-muscle cells and depends solely on the filling status of the Ca²⁺ stores (Launikonis and Rios 2007).

 SOCE modulation by RyR1 and CSQ

Ca²⁺ imaging experiments and patch-clamp recordings revealed that RyR1 is required for full SOCE activation. Indeed, ryr1^-/-ryr3^-/- derived MT have reduced SOCE elicited by Tg compared to ryr1^+/-ryr3^-/- MT (Pan, Yang et al. 2002) and the kinetic of activation as well as the amplitude of ICRAC current is decreased in RyR1 deficient myotubes (Yarotskyy and Dirksen 2012). It is important to note that Ryr1 is not required for proper triad formation meaning that RyR1 activity and/or presence itself at the triad is needed for maximal influx. Another study with C2C12 myotubes treated with azumolene that inhibits RyR1 hyperactivity or leakiness in patients with malignant hyperthermia, revealed decreased SOCE after caffeine/ryanodine dependent Ca²⁺ release but has no effect on Tg dependent SOCE (Zhao, Weisleder et al.

2006).Then RyR1 activity could activate SOCE independently of SR Ca²⁺ release.

On the contrary, the Ca²⁺ buffer protein highly expressed in skeletal muscle, calsequestrin 1 (CSQ1), has an inhibitory effect on SOCE (Shin, Pan et al. 2003, Zhao, Min et al. 2010). This effect is mediated by direct interaction between the C-terminal part of CSQ1 and STIM1, which hinders STIM1 binding to Orai1 and then Orai1 gating (Wang, Zhang et al. 2015). This mechanism could play a role in disease associated with decreased calsequestrin level and/or activity such as some form of malignant hyperthermia.

4.3.3. SOCE function

 SOCE during skeletal muscle development

It has long been known (almost 40 years according to the references) that Ca²⁺ was required to regulate muscle differentiation and especially to promote myoblast fusion into myotubes.

Demonstration of Ca²⁺requirement for proper myogenesis came in 1981 with the first observations showing that EGTA blocked the fusion process while the Ca²⁺ ionophore A23187 increased it (David, See et al. 1981). Indeed, cytoplasmic Ca²⁺ elevation was demonstrated to occur in myoblasts prior to fusion (Constantin, Cognard et al. 1996) via activation of prostaglandin E1 and acetylcholine receptor-dependent pathways (Entwistle, Zalin et al.

1988). The identification of SOCE players allowed deeper understanding of the Ca²⁺ pathways and then downstream targets. A first study with C2C12 myoblasts and myotubes, revealed STIM1 requirement for NFAT dependent MEF2 transcriptional activation during early myogenesis (Stiber, Hawkins et al. 2008). Other complementary studies from Bernheim’s group have clarified the role of the different SOCE players during in vitro human muscle differentiation. Orai1, Orai3 but not Orai2 and STIM1 are needed for the first step of human myoblasts differentiation but not cell fusion (Darbellay, Arnaudeau et al. 2010). STIM2 is also needed but does not have a specific role compared to STIM1 as silencing of either proteins can be compensated by over expression of the other to restore MEF2 and Myogenin expression level and subsequent proper myogenic differentiation. In addition to STIM and Orai proteins, TRPC1 and TRPC4 as SOCE channels are also involved essentially in the late steps of human myogenesis as silencing of either TRPC impaired myoblasts fusion into multinucleated myotubes (Antigny, Koenig et al. 2013). This phenotype is recapitulated after STIM1L silencing in myoblasts which favors the idea of TRPC channels and STIM1L being part of the same Ca²⁺

influx pathway (Antigny, Sabourin et al. 2017). The in vitro studies are consistent with results from muscle specific STIM1 KO mice. Indeed, these mice present post-natal defects in muscle maturation and growth due to impairment of several pathways involved in muscle development: ERK1-2, p38MAP, AKT, PGC1 alpha and beta, NFATc1 and NFATc3, MEF2C and PPAR gamma. Over expression of calcineurin in mSTIM1 KO myotubes restores AKT, ERK and NFAT level revealing the key role of calcineurin in STIM1 mediated muscle growth signaling pathways (Li, Finch et al. 2012). At the level of whole muscle, STIM1 importance in muscle growth is highlighted by decreased muscle weight and cross sectional area (CSA) that is a common phenotype between human patients harboring loss-of-function mutations in orai1 gene and mice model with reduced or abolished SOCE (Lacruz and Feske 2015). Other muscular defects that have been investigated more specifically in some studies have revealed implication of SOCE in fiber typing and sarcomere organization. In muscle specific Orai1 KO

mice, the fraction of oxidative slow fiber type I is decreased compared to WT in soleus muscle suggesting a role of SOCE in promoting fiber type I formation over type II (Carrell, Coppola et al. 2016). In STIM1 KO mice, features of congenital myopathy with central nucleation, decreased dystrophin staining and altered myofibrils organization (Stiber, Hawkins et al. 2008) are consistent with Volkers’ work on zebrafish embryo. Silencing of Orai1 induces skeletal muscle weakness with disorganized myofibrils that detach from the Z-line (similar to mutants of DAPC) in addition to Z-line disorganization. This phenotype detected at 48h post fertilization is even further pronounced at 72hpf in developing embryos. Then Orai1 is not required for first developmental step but rather in the maintenance of sarcomere’s organization and its adaptation upon higher force production happening after the initial events of differentiation (Volkers, Dolatabadi et al. 2012). All together these studies demonstrated a key role of SOCE in muscle development that explained muscle weakness as a common feature of human and animal models that have abolished or diminished SOCE.

 SOCE in adult skeletal muscle

The role of SOCE in adult skeletal muscle is still a matter of debate but knowledge of SOCE characteristics allows authors to reasonably exclude some potential role in muscle function.

Indeed, it is very unlikely that SOCE would play a significant role either in muscle contraction or SR refilling during single twitch. The influx rate of SOCE is too low compared to the amount of Ca²⁺ released by RyR1 upon depolarization to participate in muscle contraction (figure III.19) (Launikonis, Murphy et al. 2010). SOCE activation threshold is also far below the SR Ca²⁺ level after Ca²⁺ release during a single twitch (Cully and Launikonis 2013).

Figure III.19: Skeletal muscle sarcomere and Ca²⁺fluxes during EC coupling.

Adapted from Launikonis. B. S., et al. (2010) Schematic diagram of a mammalian skeletal muscle sarcomere showing SR (in red) and SOCE (in green) fluxes during EC coupling. Note that the arrows are drawn relative to the size of the respective fluxes. SOCE delivers Ca²⁺ to the junctional space only and cannot replace the role of SR Ca²⁺ release in activating the contractile apparatus. t-sys, t-tubule; tcSR, SR terminal cisternae; lSR, longitudinal SR.

However, an increasing number of studies have shown evidence of SOCE involvement in limiting muscle fatigue. Although fatigue being a very complex phenomenon not fully understood, SOCE would act at 2 levels 1) it would balance Ca²⁺ efflux from cytoplasm that occurs during prolonged excitation contraction because of NCX and PMCA activity and 2) as ionic conditions (such as Ca²⁺ precipitation due to increased inorganic phosphate) are altered under strenuous stimulations, SOCE would compensate the decreased ability to SR for taking up Ca²⁺ (Cully and Launikonis 2013). These considerations are consistent with muscular weakness and myopathies that characterize human patients harboring loss-of-function mutations in either orai1 or stim1 gene (Lacruz and Feske 2015). Moreover, one common feature of global and muscle specific STIM1 KO mice (Stiber, Hawkins et al. 2008, Li, Finch et al. 2012) or dominant negative Orai1 mice (Wei-Lapierre, Carrell et al. 2013) is an increase susceptibility to fatigue demonstrated by in vivo endurance test as well as ex vivo assays on muscle fibers. Measurements with muscle fibers from heterozygous Stim^-/+ or Orai1 dominant negative mice show comparable phenotype with decreased force production associated with decreased Ca²⁺ transients compared to WT upon tetanic stimulations (Stiber, Hawkins et al.

2008, Wei-Lapierre, Carrell et al. 2013). However, as mentioned previously SOCE is implicated in muscle development as humans and animals with impaired SOCE show decreased muscle weight associated in some instances with features of myopathy. In such conditions, it is difficult to assign the increased muscle weakness and susceptibility to fatigue to impaired muscle development or altered muscle function. To definitively resolve this question, Carrell and co-workers evaluated muscle force and fatigue in tamoxifen-inducible, muscle-specific Orai1 KO mice (Carrell, Coppola et al. 2016). At 4 month of age, mice were injected with tamoxifen and Orai1 transcript and SOCE efficiency assessed one month later. In these conditions, SOCE was decreased by 80% without any alteration in muscle structure (CSA, fiber type distribution, MyHC content). Interestingly, tamoxifen-injected mice behaved similarly to control during endurance tests and results from ex-vivo measurements of force production on isolated muscle were comparable between KO and WT. As the defective phenotype with increased susceptibility to fatigue identified in constitutive Orai1 KO mice was not recapitulated in adult mice after acute Orai1 deletion, it is likely that muscle fatigue associated with impaired SOCE (both in mice and human) is due to muscle developmental malformations rather than defective Ca²⁺ handling and more precisely SR refilling.