Results

Characterization of a STIM1L specific antibody

To determine STIM1L localization, we first developed and validated an antibody recognizing specifically the long isoform as so far no commercial antibody is available. Recombinant antibodies core facility of the Geneva Faculty of Medicine produced this antibody by using the specific part of the long STIM1L isoform (106 aa) as bait for the phage display technique (Blanc, Zufferey et al. 2014). To ascertain that the antibody we got recognized only STIM1L and none of the other STIM isoforms, we over-expressed YFP-tagged STIM1, YFP-STIM1L and YFP-STIM2 in DKO MEFs that are deprived of any endogenous STIM isoforms. Immunostaining with the STIM1L antibody revealed a specific signal in DKO MEFs over-expressing STIM1L but not in cells over-expressing neither STIM1 nor STIM2 (figure VII.1A). These results provided evidence of a selective recognition of exogenously expressed STIM1L over STIM1 and STIM2. Then we assessed whether this antibody recognized endogenous STIM1L protein specifically. Previous studies have shown that STIM1L expression increased during myogenic differentiation: it is not present in myoblasts and highly expressed after 48h of differentiation in myotubes (Darbellay, Arnaudeau et al. 2011, Antigny, Sabourin et al. 2017). Thus we did immunostaining in myoblasts and myotubes with the STIM1L antibody together with MEF2 antibody (that recognizes several MEF2 isoforms) as an early marker of differentiated myotubes (figure VII.2). A signal with the STIM1L antibody was detected only in myotubes and not in myoblasts that expressed STIM1 and STIM2 (Darbellay, Arnaudeau et al. 2010). This experiment confirmed that the STIM1L Ab does not recognized endogenous STIM1 nor STIM2 but very likely endogenous STIM1L. Indeed, we cannot rule out the possibility that this antibody recognizes unspecifically another protein expressed in myotubes and not in myoblasts. Eventually, we tried to use the antibody in Western blot (WB) with protein extracts from 72h differentiated myotubes (figure VII.1C). Unfortunately, we did not obtain any signal contrary to membrane blotted with a commercial STIM1 antibody that recognizes both isoforms and used as positive control in these experiments. It is possible that the STIM1L antibody detects the protein with preserved tridimensional conformation which is the case in immunostaining experiments but not after denaturation with detergent like it is done for WB.

Figure VII.1: Characterization of a STIM1L specific antibody.

A- Immunostaining with a STIM1L specific antibody and AlexaFluor 546-conjugated secondary antibody in Stim1^-/-Stim2^-/- MEFs expressing YFP-STIM1, YFP-STIM1L or YFP-STIM2. Results from n=3 independent transfections with YFP-STIM1 and YFP-STIM1L, and n=1 transfection with YFP-STIM2.

Scale bar 20 µm.

B- Co-immunostaining with DAPI (in blue) for nuclei labelling, MEF2 (in red) as a marker of differentiated myotubes and a STIM1L specific antibody (in green) in human primary myoblasts (upper panel) and myotubes after 48h in differentiation medium (bottom panel). Results from n=1 experiment. Scale bar 40 µm.

C- Western blot with 30µg of protein extracts prepared from myotubes after 72h (MT 72h) in differentiation medium and probed with a STIM1L specific antibody (left panel) or a STIM1 commercial antibody recognizing both isoforms (right panel). α-Tubulin was used as loading control. Results from n=1 experiment.

Figure VII.2: STIM1L is expressed in slow and fast muscle fibers.

A- Co-immunostanning of a transversal cryosection of hamstring muscle from a human patient. In the upper pannel, all muscle fibers are labelled in yellow (spectrin antibody), fast fibers are in red (SERCA1 antibody) and STIM1L in green. In the bottom pannel, all muscle fibers are labelled in yellow (spectrin antibody), slow fibers are in red (SERCA2 antibody) and STIM1L in green. n=4-5 independent experiments. Scale bar 100 µm.

B- Same as A except for the green labelling which shows both STIM1 and STIM1L isoforms (commercial antibody). n=2 independent experiments. Scale bar 100 µm.

STIM1L is expressed in slow and fast muscle fibers

As mentioned in the introduction, a muscle is composed of different muscle fibers classified according to their fatigue resistance (Schiaffino and Reggiani 2011). As the main phenotype of human patients and STIM1 KO mice is an increased susceptibility to fatigue (Stiber, Hawkins et al. 2008, Li, Finch et al. 2012, Lacruz and Feske 2015), we wondered whether STIM1L would be expressed preferentially in a certain type of muscle fiber conferring them an advantage in fatigue resistance. To answer this question, we did immunostaining with the STIM1L Ab on human skeletal muscle surgical waste. Tissues from surgical waste are not as well preserved as intact biopsies and can begin to deteriorate between sampling and experiment starting.

Muscle bundles from surgical waste appear dissociated in some cases as conjunctive tissue loosens up letting empty spaces appear between fibers that do not exist in the native tissue.

To distinguish between holes due to poor preservation of the sample and absence of STIM1L signal, we labelled all the fibers with an antibody against spectrin, that is a scaffolding protein linking PM and actin cytoskeleton and expressed in all muscle fibers. Comparison between spectrin labelling and STIM1L staining revealed that all muscle fibers expressed STIM1L (figure VII.2A). In combination with spectrin and STIM1L antibodies, we used either SERCA1 or SERCA2 antibodies as markers of the different fiber types: SERCA1 is specific of fast skeletal muscle while SERCA2 is solely present in slow muscle fibers (Schiaffino and Reggiani 2011).

SERCA1 and SERCA2 labelling resulted in a typical patchy pattern according to the fiber type marked while STIM1L was homogenously expressed, independently of fiber type (figure VII.2A). These results were confirmed by co-immunostaining between either SERCA1 or SERCA2, spectrin and both STIM1 isoforms with a commercial antibody (named STIM1-1L antibody) that also showed STIM1 and STIM1L presence in slow and fast muscle fibers (figure VII.2B). Given that STIM1L is homogenously expressed in all fibers (figure VII.2A) and STIM1-STIM1L staining with the commercial antibody is also homogenous (figure VII.2B), we can deduce that STIM1 isoform is also expressed with equal level in fast and slow fibers.

Obviously, this rationale supposes that the commercial antibody recognizes STIM1 and STIM1L with the same affinity, which is likely because the antibody targets the same epitope in both isoforms.

STIM1 and STIM1L are mainly expressed in the longitudinal SR

In myotubes, STIM1L interacts with cortical actin and forms permanent clusters with Orai1 channels independently of Ca²⁺ store content (Darbellay, Arnaudeau et al. 2011). To determine whether in adult muscle fibers STIM1L is also located near the PM and more specifically at the triads where Orai1 has been reported (Edwards, Murphy et al. 2010), we performed co-immunostaining with STIM1L and either RyR1 or SERCA. RyR1 is a marker of junctional SR that are in close apposition with the t-tubules to form triads (Flucher 1992) whereas SERCA1 and SERCA2 are expressed in the longitudinal SR (Jorgensen, Shen et al. 1982, Salanova, Priori et al. 2002) (See Introduction figure III.14 for further details on l-SR and j-SR staining in adult skeletal muscle). Surprisingly, RyR1 immunostaining in a double rows striation pattern characteristic of this marker, showed only partial co-localization with STIM1L (figure VII.3A). STIM1L striations were localized in between RyR1 double rows as shown in figure VII.3A and with the plot profile (figure VII.3E). Conversely, confocal images of SERCA1 (figure VII.3B, F) and SERCA2 (figure VII.3C, G) staining demonstrated typical striations every 1.5 µm that co-localizes perfectly with STIM1L signal, suggesting its presence in the longitudinal SR rather than at the terminal cisternae. Finally, to determine STIM1 distribution in adult skeletal muscle, as no specific antibody was available, we thought that if STIM1 was not localized at the longitudinal SR exclusively as previously described (Stiber, Hawkins et al.

2008, Wei-Lapierre, Carrell et al. 2013), co-localization between a STIM1 commercial antibody and STIM1L antibody would not overlap completely. On the contrary, co-immunostaining between a commercial antibody recognizing both STIM1 isoforms and the STIM1L specific antibody revealed strong co-localization (figure VII.3D, H) of the two signals. Similar experiments with another STIM1 commercial antibody, again not specific of any STIM1 isoforms, showed comparable results (data not shown). According to these results, STIM1 and STIM1L seem both localized mainly in the long part of the SR (more at the I-A band) in human adult skeletal muscle.

Figure VII.3: STIM1 and STIM1L are mainly expressed in the longitudinal SR.

(A-D)- Co-Immunostaining of longitudinal cryosection from hamstring muscle biopsies from a human patient with antibodies against STIM1L in green and in red either RyR1 (A), SERCA1 (B), SERCA2 (C) and STIM1 and STIM1L (D). (E-H) Line profile of normalized fluorescence intensity (white dotted line) of STIM1L together with either RyR1 (E), SERCA1 (F), SERCA2 (G) and STIM1 and STIM1L (H). STIM1 and STIM1L are localized preferentially in the JSR (Jonctionnal Sarcoplasmic Reticulum) as SERCA1 and SERCA2. n=2-5 independent experiments. Scale bar 2 µm.

STIM1 interacts with the Dystrophin-associated protein complex (DAPC)

It is now accepted that TRPC channels participates in SOCE in numerous cell types including muscle cells (Antigny, Koenig et al. 2013, Ong, de Souza et al. 2016). On one hand, STIM1 and STIM1L have been shown to interact with TRPC3 and TRPC6 in heterologous system (Horinouchi, Higashi et al. 2012) as well as with TRPC1 and TRPC4 in human myotubes (Antigny, Sabourin et al. 2017) and on the other hand, TRPC1 and TRPC4 are associated with the DAPC via interaction with the PDZ domain (Post synaptic density protein, Drosophila disc large tumor suppressor, and Zonula occludens-1 protein) of α1-syntrophin, a signaling protein of the DAPC (Vandebrouck, Sabourin et al. 2007, Sabourin, Lamiche et al. 2009). Knowing that, we wondered whether STIM1 and/or STIM1L could interact directly or indirectly with the DAPC in human skeletal muscle. To answer this question, we performed co-immunoprecipitations with the STIM1L specific antibody but we failed to obtain a signal in such experiments (data not shown). Then we used a STIM1 commercial antibody (figure VII.4A) or a dystrophin antibody (figure VII.4B) to immuno-precipitate human skeletal muscle extracts. Incubation of the extracts with magnetic beads was used as negative control whereas the presence in the immuno-precipitated complexes of the target protein was checked as positive control for each IP. Immunoblot against STIM1 and STIM1L confirms that both proteins were immuno-precipitated with modest enrichment compared to the amount of proteins in the lysate (that represents only 10% of the total extract used for the IP) (figure VII.4A, bottom panel). The upper panel of figure VII.4A demonstrates the presence of dystrophin in the STIM1 IP, confirming an interaction between this protein and STIM1 and/or STIM1L. Similarly, dystrophin detection by immunoblot after dystrophin IP (figure VII.4B, upper panel) ensured the proper immuno-precipitation of the protein and immunoblot with STIM1 antibody confirmed interaction with STIM1 and possibly STIM1L (asterisk in figure VII.4B, bottom panel). To note a slight shift of STIM1 migration in dystrophin IP in regard with the lysate due to non-homogenous migration. More importantly, the quantity of protein immuno-precipitated was significantly higher for STIM1 than for STIM1L while the respective amount of both protein was similar in the lysate. This indicates a preferential interaction between STIM1 and dystrophin compared with STIM1L and dystrophin. These results are consistent with the respective localization of the 3 proteins in adult skeletal muscle. In muscle fibers, dystrophin immunostaining shows pattern corresponding to the Z-line as the DAPC is

part of the costamere which constitutes a mechanical link between the sarcolemma and the sarcomeres (via the Z-line) (Rybakova, Patel et al. 2000). Then we did co-immunostaining between a Z-line marker, α-actinin, and STIM1-1L (with the commercial antibody) (figure VII.4C). Resulting images demonstrate partial overlapping signal between the two staining consistently with STIM1 and STIM1L localization along the longitudinal SR, in line with the Z-line (see Fig13 of the introduction) as attested by with co-immunostaining with RyR1 antibody (figure VII.4D). Plot profile of α-actinin and RyR1 fluorescence intensity confirmed the images (figure VII.4E-F). To note, α-actinin plot profile was narrowed compared to STIM1-1L signal that was around 0.2-0.3 µm larger because of α-actinin is restricted to the Z-line while the long SR extends over this structure. All together these results point toward a direct or more likely indirect interaction between the DAPC and STIM1 and possibly STIM1L.

Figure VII.4: STIM1 and STIM1L interact with the DAPC.

(A-B)- Representative co-immunoprecipitation experiments in human myotubes. Lysates from human muscle were incubated with antibodies against dystrophin (A), STIM1 commercial antibody recognizing both isoforms (B) or magnetic beads only as negative control. Western blots of the immunoprecipitated proteins were probed with antibodies against dystrophin or STIM1 commercial antibody as indicated on the right. These results are representative of two independent experiments performed with human muscle samples from different patients.

(C-D)- Co-Immunostaining of longitudinal cryosection from hamstring muscle biopsies human patient with antibodies against both STIM1 isoforms in green and in red either α-actinin (C) or RyR1 (D). n=1 experiment. Scale bar 2 µm.

(E-F)- Line profile of normalized fluorescence intensity (white dotted line) of both STIM1 isoforms together with either α-actinin (E) or RyR1 (F).

Expression and localization of EC coupling associated proteins in human myofibers after 2 weeks of in vitro differentiation

In order to study STIM1L function in human muscle physiology, we decided to develop a model of in vitro differentiated muscle fibers derived from human satellite cells taken from healthy patients subjected to orthopedic surgery for cruciate ligament injuries. The initial purpose was to silence STIM1 or STIM1L in human muscle fibers obtained with such model and study consequences in terms of Ca²⁺ fluxes upon SR depletion in the cytosol as well as in SR and mitochondria. To develop this model we decided to combined methods we already used to isolate human satellite cells and differentiate them in vitro (Laumonier, Koenig et al. 2017) with a method described by Falcone and colleagues that was devised to get muscle fibers with mature EC coupling machinery after 10 days of differentiation from mice satellite cells (Falcone, Roman et al. 2014) (figure VII.5A). The advantage of Falcone and co-workers’ model is the use of a matrigel layer above myotubes which avoids their detachment when they contract. Indeed, this was one the main problem encounters when we cultured myotubes that prevented us to keep them a long time in culture in order to reach the maturation step desired. After 15 days in differentiation medium (DM), we evaluated the expression level of several Ca²⁺ handling proteins as well as the 3 adult MyHC isoforms by Real Time PCR (RT-PCR) to determine the differentiation status of the myofibers. Figure VII.5B shows the relative mRNA level on a logarithmic scale of the proliferating myoblasts and myofibers compared to adult muscle sample for the genes chosen. Transcript level of MYH1, MYH2 and MYH7 corresponding to MyHC-2X, MyHC-2A and MyHC-β/slow proteins expressed in fast 2X, fast 2A and slow fibers respectively increased during the differentiation but are still around 10 fold lower in myofibers than in adult muscle. Similarly, we noted an augmentation of the expression level of the two triadic markers that are DHPR and RyR1 in myofibers compared to myoblast. However, DHPR and RyR mRNA level are 3 and 2 fold less abundant respectively in differentiated muscle fibers than in adult biopsies. Surprisingly, the amount of SERCA1 and SERCA2 transcripts do not increase during the differentiation and are around 10 times for SERCA1 and 100 times for SERCA2 lower in culture than in adult samples. Finally, STIM1 transcript level do not vary between the conditions tested while the quantity of STIM1L mRNA in myofibers is comparable to adult muscle. All together these results indicate an intermediate state of differentiation reached by the myofibers after 15 days in culture.

As described in the introduction, adult skeletal muscle possesses a very peculiar architecture dedicated to couple excitation and contraction. Localization of proteins implicated in EC coupling determines the differentiation level of myofibers as they progressively organized in relation to one another. Thus we performed immunostainings to evaluate the level of maturation and organization of the myofilaments, z-lines, l-SR and j-SR with antibodies against MyHC isoforms, α-actinin, SERCA2 and RyR1 respectively (figure VII.5C). MyHC and α-actinin showed nice striation pattern, regularly spaced by 2.5μm demonstrating the formation of mature sarcomeres in our culture, in accordance with spontaneous contractions seen in the petri dish for several myofibers (data not shown). L-SR visualized by SERCA labelling also showed striations spaced by 2.5μm that illustrate a l-SR in place. However, RyR1 staining showed longitudinal lines which denoted j-SR/terminal cisternae presenting longitudinal orientation and not the typical double row pattern when triads are fully mature. Then, consistent with mRNA results, myofibers obtained after 15 days of differentiation recapitulated some characteristics of adult muscle with fully-formed sarcomeres and l-SR but not completely mature EC coupling as triads were not in place.

Figure VII.5: Expression and localization of EC coupling associated proteins in human myofibers after 2 weeks of in vitro differentiation.

(A)- qPCR results of the relative expression levels of the indicated transcripts in myoblasts, 15 days differentiated myofibers and adult muscle. Expression levels were normalized for 2 housekeeping genes (β2 microglobulin, ALAS1) and expressed as relative to adult transcript content.

(B)- On the top, immunostanning of human myofibers obtained after 15 days in culture with several markers: MHC, α-actinin, SERCA2 and RyR1 to assess the differentiation level of myofibrils, z-lines, l-SR and j-l-SR/terminal cisternae respectively. On the bottom, line profile of fluorescence intensity (white dotted line) of the corresponding immunostaining on the top panels. Scale bar 5 µm.

Trials to obtain in vitro differentiated human myofibers with mature triads

Because STIM1 and STIM1L are not evenly distributed in the SR as visualized in adult human sample (figure VII.3) and can potentially translocate from one part to another during SOCE induction, we thought it was important to study their function in relation to their localization in myofibers presenting mature SR similar to adult sample. As SR is composed of l-SR and j-SR and we failed to obtained j-SR forming triads with proper longitudinal orientation during the first tests, we decided to try out a number of different conditions to improve our model.

Several parameters can be changed to improve the level of differentiation and we decided to focus on 3 of them based on literature as well as for practical reasons: DM composition, duration time in DM and composition of cell mixture to differentiate. Figure VII.6A summarizes the conditions tested and the results we obtained in terms of triads formation with transverse orientation assessed by immunostaining with RyR1 antibody. According to several studies, we modified the composition of the DM by either adding alone or in combination different compounds that are: dexamethasone (Syverud, VanDusen et al. 2016), rat or human agrin, horse or fetal calf serum (Falcone, Roman et al. 2014) to the DM used classically in the lab. We also tried to change the base for preparing DM with IMDM instead of DMEM (Falcone, Roman et al. 2014) but none of these conditions gave us the results expected (figure VII.6A, line 1-7).

Thus, as triads formation is known to be a long process that terminates only after birth during mouse embryogenesis (Takekura, Flucher et al. 2001), we decided to let the myofibers a longer time in culture, up to 6 weeks in DM with or without some of the additional compounds previously cited. Unfortunately, these trials were not successful as well (figure VII.6A, line 8-14). Finally, as the few studies describing triads formation in vitro were realized from a mixture of mice or rat muscle cells obtained directly after dissociation without cell sorting by flow cytometry (Cusimano, Pampinella et al. 2009, Falcone, Roman et al. 2014), we also tested this procedure additionally to the other conditions tested (figure VII.6A, line 15-16). Interestingly, mixture of cell population let 4 weeks in culture with DM containing rat agrin during the first 2 weeks that was removed for the last 2 weeks, differentiated into myofibers with triads showing a beginning of transverse orientation (figure VII.6B). To note that after 2 weeks of differentiation, no triads were formed in the similar condition (figure VII.6A, line 15) indicating that not only the cell type present in the petri dish but also the duration in culture are both crucial parameters. However, the successful condition was tested only once as the following

trials to reproduce it failed due to detachment of the cellular layer before 4 weeks of differentiation. Improvement of the method to keep myofibers a long time in culture and/or fasten the differentiation are crucial to reproduce this promising result.

Figure VII.6: Trials to obtain in vitro differentiated human myofiber with mature triads.

Figure VII.6: Trials to obtain in vitro differentiated human myofiber with mature triads.