ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.

TAM and STRMK (OMIM #160565 and #615883) form a clinical continuum, and the occurrence and degree of the pathologic signs depend on the implicated gene and the position of the mutation.The majority of patients with TAM/STRMK carry heterozygous missense mutations in STIM1, encoding a ubiquitously expressed Ca 2+ sensor residing at the endoplasmic/sarcoplasmic reticulum (ER/SR).Mutations in the Ca 2+ -sensing EF-hand in the luminal part of STIM1 essentially give rise to a muscle phenotype with occasional depletion of platelets and anomalies of skin, spleen, and bones (5)(6)(7)(8)(9)(10)(11)(12)(13), while mutations in the cytosolic coiled-coil domain and more specifically of the arginine 304 residue correlate with a multisystemic presentation of the disorder (8,10,(14)(15)(16)(17)(18)(19).Missense mutations in the plasma membrane Ca 2+ channel ORAI1 are less common and either affect the pore-forming transmembrane domain and cause a severe phenotype with marked muscle weakness, miosis, bleeding diathesis, ichthyosis, and Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism.TAM/ STRMK is caused by gain-of-function mutations in the Ca 2+ sensor STIM1 or the Ca 2+ channel ORAI1, both of which regulate Ca 2+ homeostasis through the ubiquitous store-operated Ca 2+ entry (SOCE) mechanism.Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca 2+ .There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation.Here, we crossed Stim1 R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1 R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca 2+ influx.Compared with Stim1 R304W/+ littermates, Stim1 R304W/+ Orai1 R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics.Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1 R304W/+ muscle and revealed a major restoration of gene expression in Stim1 R304W/+ Orai1 R93W/+ mice.Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.

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JCI Insight 2024;9(6):e174866 https://doi.org/10.1172/jci.insight.174866dyslexia or induce amino acid substitutions in the concentric channel rings and give rise to a more moderate phenotype (18,(20)(21)(22).Finally, missense mutations in the muscle-specific SR Ca 2+ -buffering protein calsequestrin (CASQ1) and in the muscle-specific SR Ca 2+ release channel RyR1 form the mild end of the TAM/STRMK spectrum and were found in patients with adult-onset muscle weakness in the absence of multisystemic signs (23)(24)(25).
Ca 2+ is essentially stored in the ER/SR and serves as a ubiquitous and highly versatile second messenger in all eukaryotes.In response to external stimuli, Ca 2+ ions are temporarily and spatially released to the cytoplasm where they induce various signaling cascades and exert a plethora of biological processes, including proliferation, neuronal transmission, hormone secretion, or coagulation (26).In myofibers, Ca 2+ is bound to calsequestrin polymers in the SR and is released through RyR1, and the increase of myoplasmic Ca 2+ concentrations initiates muscle contraction (27).As a consequence of Ca 2+ store depletion in both excitable and nonexcitable cells, STIM1 undergoes a conformational change and assembles into oligomers able to activate ORAI1 and trigger extracellular Ca 2+ entry (28,29).This fundamental mechanism of Ca 2+ store refilling, known as store-operated Ca 2+ entry (SOCE), ensures the maintenance of high intracellular Ca 2+ gradients for oscillatory Ca 2+ signaling.
There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation, and the availability of a faithful animal model offers the possibility of assessing therapeutic approaches.As the most downstream SOCE component, the Ca 2+ channel ORAI1 constitutes the principle target for a common therapy for both STIM1-and ORAI1-related TAM/STRMK.To provide the experimental evidence that TAM/ STRMK can be prevented by the targeted downregulation of Orai1, we previously crossed Stim1 R304W/+ mice with Orai1 +/-mice, and the Stim1 R304W/+ Orai1 +/-offspring carrying the recurrent TAM/STRMK mutation and expressing only 50% of ORAI1 underwent extensive phenotyping (41).In contrast to Stim1 R304W/+ littermates, Stim1 R304W/+ Orai1 +/-mice were born with the expected Mendelian birth ratio and showed significantly increased postnatal growth and bone architecture and partially improved muscle contraction and relaxation parameters (41).However, the platelet and spleen phenotypes were comparable in Stim1 R304W/+ Orai1 +/-and Stim1 R304W/+ mice, exemplifying the incomplete rescue of the multisystemic TAM/STRMK phenotype via ORAI1 downregulation and demonstrating the need for alternative and more efficient therapeutic strategies.
Overall, this work provides a therapeutic proof of concept and illustrates that the inhibition of ORAI1 activity is a potent method to anticipate the multisystemic signs characterizing TAM/STRMK.

Results
To date, 4 murine models with different STIM1 GoF mutations in the luminal EF-hands or in the cytosolic coiled-coil domains have been described (33,35,38,42), but only the Stim1 R304W/+ mouse harboring the most common TAM/STRMK mutation recapitulates the multisystemic signs of the human disorder (35).Indeed, Stim1 R304W/+ mice are smaller than WT littermates, manifest reduced muscle strength, prolonged bleeding times, skin irritations, and spleen anomalies (35) and, thus, represent the most suitable animal model to assess therapeutic approaches.
To follow early postnatal development, the offspring underwent regular measurements of body size and weight over the first 4 months.At every time point, Stim1 R304W/+ mice were significantly smaller and lighter than WT and Orai1 R93W/+ controls, confirming our previous observations (35).In contrast, Stim1 R304W/+ Orai1 R93W/+ mice showed continuously higher growth curves, with an increase of body weight and length at 4 months of age compared with Stim1 R304W/+ mice (Figure 1A and Supplemental Figure 2A).To investigate the factors contributing to the growth disparities, we examined bone morphology by micro-computerized tomography.By contrast with those from Stim1 R304W/+ mice, femurs from Stim1 R304W/+ Orai1 R93W/+ mice showed a normal trabecular thickness and bone marrow density, and the overall trabecular structure was comparable with healthy WT and Orai1 R93W/+ controls, highlighting a rescue of bone architecture through ORAI1 inhibition (Figure 1B and Supplemental Table 2).
Megakaryocytes generate and release platelets into the bloodstream, where they play a pivotal role in thrombus formation in response to vessel wall damage (43).Low platelet numbers in combination with bleeding diathesis is a major clinical feature of TAM/STRMK (2,7,8,10,11,(14)(15)(16)(17)(18)(19)(20)44), and studies in murine Stim1 models have shown that platelet preactivation and increased turnover account for the decline of thrombocytes (35,42).The peripheral blood of Stim1 R304W/+ mice contained only 25% of the normal platelet quantity, and the number of circulating platelets was more than doubled in Stim1 R304W/+ Orai1 R93W/+ mice (Figure 1F).Accordingly, in vivo assessment of platelet function revealed excessive bleeding times in Stim1 R304W/+ mice and a significant improvement in Stim1 R304W/+ Orai1 R93W/+ littermates (Supplemental Figure 2B).Although WT values are not reached, these data support a relevant effect of ORAI1 inhibition on the thrombocytopenia phenotype.
Improved muscle performance and physiology.Muscle weakness and exercise intolerance constitute the primary clinical signs of TAM/STRMK (3).While the age of onset, disease course, and severity can vary among and between families, first symptoms commonly occur during childhood or adolescence (2).Like many patients, Stim1 R304W/+ mice are phenotypically normal at birth and manifest a loss of general and specific muscle force before reaching adulthood (35).
To assess the effect of ORAI1 inhibition on muscle performance, our mice underwent hanging, openfield, and force transduction tests.Between 1 and 4 months of age, the ability of Stim1 R304W/+ mice to hang upside down on a cage grid gradually dropped down to 50% of the normal values, while the performance of Stim1 R304W/+ Orai1 R93W/+ littermates remained largely stable with values around 75% of the WT reference

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JCI Insight 2024;9(6):e174866 https://doi.org/10.1172/jci.insight.174866(Figure 2A).In line with the augmented general muscle force, Stim1 R304W/+ Orai1 R93W/+ mice covered a longer distance with a higher velocity than Stim1 R304W/+ littermates in the open-field arena at 10 weeks of age (Supplemental Figure 2, C and D).
We next assessed the in situ muscle contraction and relaxation properties on anesthetized animals through electrical stimulation of the fast-twitch tibialis anterior (TA) muscle.A previous report found higher Orai1 expression levels in fast-twitch type II than in slow-twitch type I myofibers in mice (45), suggesting that the TA is an appropriate muscle to detect a potential therapeutic effect of ORAI1 inhibition on muscle function.At low stimulation frequencies between 10 and 40 Hz, the force of Stim1 R304W/+ muscles increased more rapidly compared with healthy WT and Orai1 R93W/+ controls (Figure 2B).In addition to premature muscle contraction, Stim1 R304W/+ mice also showed a significant delay in muscle relaxation following single (Figure 2, C and D) and tetanic (Figure 2, E and F) stimulations.Indeed, the relaxation time of Stim1 R304W/+ muscle was twice as long as that in the controls and resulted in abnormal fatigue curves (Supplemental Figure 2, E  and F).Compared with Stim1 R304W/+ littermates, muscle contraction at low stimulation frequencies was normalized in Stim1 R304W/+ Orai1 R93W/+ mice (Figure 2B) and muscle relaxation was significantly improved (Figure 2, C-F), demonstrating a positive effect of ORAI1 inhibition on muscle functionality.
To decipher the physiological events leading to normal or pathologic muscle contractility, we isolated primary myoblasts from WT, Orai1 R93W/+ , Stim1 R304W/+ , and Stim1 R304W/+ Orai1 R93W/+ mice and differentiated the mononuclear cells into myotubes.Quantification of the cytosolic Ca 2+ content and extracellular Ca 2+ entry revealed a substantial elevation of resting Ca 2+ and SOCE amplitude in Stim1 R304W/+ myotubes compared with WT and Orai1 R93W/+ controls (Figure 2, G and H), possibly providing a molecular link with the abnormal muscle contraction and relaxation dynamics in Stim1 R304W/+ mice.Accordingly, the decrease of resting Ca 2+ levels and SOCE amplitude in Stim1 R304W/+ Orai1 R93W/+ myotubes compared with Stim1 R304W/+ myotubes (Figure 2, G and H) correlated with the normalization of muscle contraction and the improvement of muscle relaxation in Stim1 R304W/+ Orai1 R93W/+ mice (Figure 2, B-F).
Normalized muscle morphology and muscle degeneration markers.Complementary to the in vivo and in situ muscle force experiments, we dissected the fast-twitch TA and the mixed gastrocnemius muscle of WT, Orai1 R93W/+ , Stim1 R304W/+ , and Stim1 R304W/+ Orai1 R93W/+ mice to examine muscle morphology.
Histological analyses of Stim1 R304W/+ TA and gastrocnemius samples showed myofiber atrophy, abnormal nuclear internalization, and the infiltration of immune cells, all hallmarks of myofiber degeneration (Figure 3, A and B, and Supplemental Figure 3, A and B).In contrast, muscle sections from Stim1 R304W/+ Orai1 R93W/+ mice did not show any difference compared with WT mice and no signs of inflammation and myofiber degeneration, highlighting the rescue of skeletal muscle integrity.
In Stim1 R304W/+ mice, myofiber degeneration is accompanied by elevated serum creatine kinase (CK) levels and enhanced regeneration, as illustrated by an increased number of fibers expressing embryonic myosin heavy chain (eMHC) (35).In contrast, CK levels in most Stim1 R304W/+ Orai1 R93W/+ blood samples were within normal ranges (100-200 U/L) (Figure 3C), and immunofluorescence experiments on Stim1 R304W/+ Orai1 R93W/+ muscle sections showed a normal ratio of eMHC-positive myofibers (Figure 3, A and D), confirming the absence of enhanced myofiber degeneration and regeneration cycles.This is also supported by the analysis of unfolded protein response (UPR) markers.We previously showed that the increased cytosolic Ca 2+ levels in Stim1 R304W/+ muscle induce reticular Ca 2+ stress and trigger UPR, which ultimately leads to myofiber degeneration (40).Quantification of selected UPR markers in TA muscle extracts confirmed an increased expression of the chaperone genes Hspa5 and Hsp90b1 and an increased ratio of spliced versus unspliced Xbp1 in Stim1 R304W/+ mice and showed a normalization of the expression levels and splicing ratio in Stim1 R304W/+ Orai1 R93W/+ littermates (Figure 3E), demonstrating that Ca 2+ stress was fully resolved.
ORAI1 inhibition is more efficient than Orai1 downregulation.As the first therapeutic strategy to treat TAM/ STRMK, we previously investigated the potential of Orai1 downregulation, and the systematic characterization of Stim1 R304W/+ Orai1 +/-mice revealed an amelioration of several but not all phenotypes affecting bones, skeletal muscle, spleen, and platelets (41).Here, we tested a different approach, and the genetic inhibition of the Ca 2+ channel ORAI1 substantially improved or rescued all signs of the multisystemic TAM/ STRMK phenotype in the Stim1 R304W/+ mouse model.
To determine the benefit of ORAI1 inhibition and Orai1 downregulation on TAM/STRMK at the molecular level, we performed RNA-Seq on WT, Stim1 R304W/+ , Orai1 R93W/+ , Stim1 R304W/+ Orai1 R93W/+ , and Stim1 R304W/+ Orai1 +/-TA muscle samples (Figure 4B).Hierarchical clustering of the sequencing data revealed a separate sample grouping of WT and Stim1 R304W/+ samples, with an upregulation or downregulation of more than 1,200 genes in the TAM/STRMK mouse model compared with the WT.The Stim1 R304W/+ and Stim1 R304W/+ Orai1 +/-samples largely clustered together and revealed a rescue of only 1.8% of the dysregulated genes, while the expression of 7.5% of the genes at least partially shifted toward the WT values (Figure 4C).In contrast, Stim1 R304W/+ Orai1 R93W/+ mice widely clustered with the healthy WT and Orai1 R93W/+ controls.The full rescue of 57.3% of the genes and the partial normalization of 22.2% of the genes attest the significantly higher therapeutic effect of ORAI1 inhibition compared with Orai1 downregulation.
To retrieve the functional profile of the normalized genes and to define the pathways implicated in the phenotypic rescue of the TAM/STRMK mice, we performed gene ontology term enrichment analyses.The Stim1 R304W/+ Orai1 R93W/+ and Stim1 R304W/+ Orai1 +/-mice shared 113 rescued genes with an enrichment of gene ontology terms associated with muscle differentiation and contraction (Figure 4D and Supplemental Figure 5).As the main difference between both therapy cohorts, only the Stim1 R304W/+ Orai1 R93W/+ mice displayed a rescue of genes implicated in ER/SR stress and immune response (Supplemental Figure 5B).This is of particular interest since myofiber degeneration involves immune cells to mediate muscle fiber clearance (46) and emphasizes the protective effect of ORAI1 inhibition against TAM/STRMK-typical myofiber degeneration and regeneration (40).Of note, the Stim1 R304W/+ Orai1 R93W/+ muscle samples featured a normalized expression of Atp2a1 and Sln, encoding the Ca 2+ pump SERCA and the negative SERCA regulator sarcolipin, respectively (Figure 4E).These data indicate that Ca 2+ may be more efficiently removed from the cytosol, which presumably contributes to the improved muscle relaxation of Stim1 R304W/+ Orai1 R93W/+ myofibers (Figure 2, C-F).
Myostatin as circulating biomarker in TAM/STRMK mice and patients.Circulating biomarkers are of major medical importance for following disease progression and evaluating therapeutic efficiencies in noninvasive or minimally invasive ways, and they take a central role in clinical trials for neuromuscular disorders (47).It has previously been shown that CK levels are increased 10-fold in patients with TAM/STRMK and mice and correlate with myofiber degeneration (2,33,35), and here, we describe a significant reduction of the serum CK levels in Stim1 R304W/+ Orai1 R93W/+ mice (Figure 3C).To complement the CK measurements and to provide an additional circulating biomarker for TAM/STRMK, we compared the list of dysregulated and rescued genes in our RNA-Seq data from WT, Stim1 R304W/+ , and Stim1 R304W/+ Orai1 R93W/+ mice, and we focused on genes expressed in skeletal muscle and coding for proteins secreted into the bloodstream and detectable in the sera of 4-month-old WT mice (48,49).Actc1, Alad, Anxa2, Clic4, Gpx1, Mstn, Sod3, and Thbs1 were all differentially expressed in Stim1 R304W/+ muscles compared with the WT and Orai1 R93W/+ controls, and the vast majority was normalized in Stim1 R304W/+ Orai1 R93W/+ mice (Supplemental Figure 6).Owing to its role as major regulator of muscle growth and its utility as biomarker in diverse myopathies (50-52), we selected myostatin (Mstn).Using an ELISA test, the level of circulating myostatin was significantly decreased in Stim1 R304W/+ plasma compared with that of healthy controls and normalized in Stim1 R304W/+ Orai1 R93W/+ samples (Figure 5A).To assess the translational potential of our findings, we next applied the ELISA test to patients with TAM/STRMK carrying different STIM1 mutations (H72Q, I115F, R304W) (Figure 5B).In all 3 patients, the myostatin levels were significantly decreased, suggesting that myostatin may serve as a suitable biomarker for TAM/STRMK in both humans and mice.

Discussion
TAM and STRMK are spectra of the same multisystemic disorder affecting skeletal muscle, bones, spleen, and platelets.No therapy is available for TAM/STRMK to date, and the absence of a treatment represents an important burden for the affected families and an unmet medical need.TAM/STRMK is caused by excessive extracellular Ca 2+ influx, and the genetic downregulation of the plasma membrane Ca 2+ channel ORAI1 in Stim1 R304W/+ Orai1 +/-mice only partially improved the multisystemic disease signs (41), pointing out the necessity for alternative therapeutic strategies.Here, we provided functional, physiological, structural, biochemical, and molecular data demonstrating that the inhibition of ORAI1

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JCI Insight 2024;9(6):e174866 https://doi.org/10.1172/jci.insight.174866significantly reduced Ca 2+ influx and increased postnatal weight gain, muscle force, and platelet numbers in Stim1 R304W/+ Orai1 R93W/+ mice, and fully rescued birth ratio, bone architecture, spleen histology, as well as myofiber morphology and turnover.
ORAI1 inhibition versus Orai1 downregulation -a measurable difference in efficacy.The Ca 2+ channel ORAI1 operates downstream of the Ca 2+ sensor STIM1 and, thus, constitutes the prime target for therapeutic approaches for the main TAM/STRMK forms -either through the regulation of its expression or through the regulation of its activity.
We assessed the therapeutic potential of both strategies in our murine Stim1 R304W/+ model and systematically examined the phenotype of Stim1 R304W/+ Orai1 +/-mice expressing 50% ORAI1 (41) and of Stim1 R304W/+ Orai1 R93W/+ mice expressing an ORAI1 mutant with constricted pore (this study).Both Stim1 R304W/+ Orai1 +/-and Stim1 R304W/+ Orai1 R93W/+ mice showed a normalized birth ratio, an improvement of trabecular bone structure associated with higher growth curves compared with Stim1 R304W/+ littermates, and an amelioration of muscle function and structure.However, the muscle contraction and relaxation dynamics were only moderately amended in Stim1 R304W/+ Orai1 +/-mice (41) and almost rectified in Stim1 R304W/+ Orai1 R93W/+ mice, indicating a superior therapeutic efficiency of ORAI1 inhibition over Orai1 downregulation.This is furthermore supported by the higher general muscle force of Stim1 R304W/+ Orai1 R93W/+ mice, the absence of enhanced SR stress and myofiber degeneration, the important reduction of cytosolic Ca 2+ levels, and the transcriptional normalization of numerous dysregulated genes in skeletal muscle.Moreover, none of the Stim1 R304W/+ Orai1 R93W/+ mice exhibited splenomegaly or an abnormal spleen histology, and all displayed a marked increase of thrombocytes compared with Stim1 R304W/+ and Stim1 R304W/+ Orai1 +/-mice.Taken together, our findings illustrate that ORAI1 inhibition resolves the multisystemic TAM/STRMK phenotype to a substantially higher degree compared with Orai1 downregulation and point to pharmacological treatment options targeting ORAI1 conduction and lowering Ca 2+ influx for prospective clinical trials.
In this context, the biphenyl-triazole CIC-39 has recently been described as a SOCE inhibitor and efficiently reduced extracellular Ca 2+ entry in fibroblasts derived from patients with TAM/STRMK (53).The CIC-39 treatment of Stim1 I115F/+ mice, exhibiting an incomplete TAM/STRMK phenotype, restored the quantity of circulating platelets and minimized bleeding diathesis (54), sustaining the idea that a steric hindrance of SOCE and ORAI1 through pharmacological compounds likely represent the most promising way to treat TAM/STRMK.
ORAI1 inhibition versus Orai1 downregulation -a different physiological effect.The ORAI1 Ca 2+ channel works as a hexamer, and each ORAI1 subunit is composed of 4 α-helical transmembrane domains, with M1 constituting the channel pore and M2-M4 shaping concentric rings surrounding the pore (55)(56)(57).In Stim1 R304W/+ Orai1 +/-mice, the 50% reduction of available ORAI1 monomers to form functional Ca 2+ channels mitigated the pathogenic impact of SOCE overactivity in the affected tissues, but only partially improved the skeletal muscle phenotype and turned out to be ineffective in spleen and platelets (41).It is possible that most or all remaining ORAI1 hexamers in Stim1 R304W/+ Orai1 +/-mice are exposed to constitutive activation through the STIM1 R304W mutant, which may counteract the therapeutic effect of Orai1 downregulation and explain the incomplete rescue.The disparate improvement levels of the affected Stim1 R304W/+ Orai1 +/-tissues possibly reflect a different Ca 2+ sensitivity and suggest that, especially, spleen cells and the spleen-derived thrombocytes may require a more stringent control of Ca 2+ balance compared with bone or skeletal muscle.
Targeting ORAI1 in other Ca 2+ -related diseases.SOCE overactivation resulting in excessive extracellular Ca 2+ entry is also reported in other muscle disorders.As an example, the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy shows high Orai1 expression levels associated with increased SOCE activity (59).And malignant hyperthermia (MH), characterized by a life-threatening sensitivity to halogenated anesthetics and caused by Ca 2+ leakage from the SR (60), implies sustained SOCE activation presumably amplifying disease severity (61).Remarkably, the exogenous expression of a dominant-negative ORAI1 mutant reduced the dystrophic features in mdx muscle samples (62,63) and the cytoplasmic Ca 2+ concentrations in MH mice-derived muscle cells (64).Similarly, treatment of murine MH cells with the unspecific SOCE inhibitors BTP-2, Gd 3+ , or GsMTx-4 decreased Ca 2+ leakage and cytosolic Ca 2+ levels in skeletal muscle (64), indicating that the therapeutic potential of ORAI1 inhibition is not restricted to TAM/STRMK.
It is debatable whether compounds specifically targeting ORAI1 or indistinctively all 3 ORAI paralogs represent the most promising strategy since a broader pharmacological range can come along with an increased risk of undesirable side effects.This is probably disorder dependent.Our results on TAM/STRMK mice suggest that the selective inhibition of ORAI1 function is sufficient to antagonize and rescue most disease signs.However, our experiments have been performed on mice with antenatal expression of the ORAI1 R93W mutation and describe the efficiency of ORAI1 inhibition to anticipate disease development.Whether the postnatal administration of ORAI1-specific molecules can effectively attenuate disease progression or revert disease signs remains to be determined.
Considering that ER stress is a major contributor to the muscle phenotype in Stim1 R304W/+ mice, any molecule modulating UPR may constitute an alternative therapeutic avenue for TAM/STRMK.As an example, treatment with the chemical chaperone 4-PBA reduced reticular stress and improved skeletal muscle function in mouse models for central core disease and Duchenne muscular dystrophy (81,82), two disorders involving a similar cellular Ca 2+ overload as TAM/STRMK.
Concluding remarks.The present study provides the proof of concept that the inhibition of the ORAI1 Ca 2+ channel improves and widely rescues the multisystemic phenotype in Stim1 R304W/+ mice, validating ORAI1 as the principal target for the treatment of TAM/STRMK.Small molecules inhibiting SOCE are currently in clinical trials for diverse human diseases, and their verification in Stim1 R304W/+ mice might accelerate their accessibility to patients with TAM/STRMK, for which the therapeutic efficacy could be monitored by the circulating biomarker myostatin.Inversely, the discovery of pharmacological compounds antagonizing TAM/ STRMK in our Stim1 R304W/+ model may also be of medical interest for other Ca 2+ -related disorders.

Methods
Sex as biological variable.With except ion of the bleeding test, all experiments were conducted on male mice.Stim1 R304W/+ male mice show an overall stronger and less variable phenotype compared with Stim1 R304W/+ female mice.
Hanging and open-field tests.To assess general muscle force, mice were suspended upside down on a cage grid for a maximum of 60 seconds, and the latency to fall was recorded.The tests were performed monthly and in triplicate with a 5-to 10-minute rest interval.The open-field test was performed on 10-week-old mice in a homogenously illuminated (100 lux at arena level) and noise-isolated room.The animals were placed in the arena (Bioseb), and rearing, velocity, and covered distance were quantified over 30 minutes.
In situ muscle force.To determine maximal and specific muscle force, 4-month-old mice were anesthetized by intraperitoneal injections of a mixture of domitor/fentanyl (2/0.28 mg/Kg), diazepam (8 mg/ Kg), and fentanyl (0.28 mg/Kg).The TA was partially excised, and the proximal tendon was attached to an isometric transducer (1305A whole animal system, Aurora Scientific).Maximal force was assessed by sciatic nerve stimulations of 1-200 Hz pulses, spaced by 30 seconds, and fatigue by 80 stimulations of 40 Hz, spaced by 2 seconds.Specific force was determined by dividing the maximal force by the muscle cross sectional area calculated as wet muscle (mg)/optimal muscle length (mm) × mammalian muscle density (1.06 mg/mm 3 ).Contraction time corresponds to the time span until maximal muscle force (100%) was reached after single stimulations.Relaxation time reflects the duration of muscle force decrease by 50% after single or tetanic stimulations.
Statistics.All cell and animal experiments were performed and analyzed in a blinded manner, and the investigators were unaware of the genotype.Normal data distribution was assessed using the Shapiro-Wilk or Kolmogorov-Smirnov test and presented as mean ± SEM.For normally distributed data, we used the 1-way ANOVA followed by Tukey's post hoc test.Otherwise, the Kruskal-Wallis followed by Dunn's multiple comparison test was used.The significance of birth ratio was determined by a χ 2 test, and the significance of myostatin levels was determined by a parametric 2-tailed t test.For body weight, hanging time, and force-frequency studies, the 2-way ANOVA followed by Tukey's post hoc test was used.P values of less than 0.05 were considered significant.
Study approval.Animal care and experimentation was in accordance with French and European legislation and approved by the Com'Eth ICS-IGBMC institutional ethics committee (Illkirch, France) and validated by the French Ministry of Higher Education and Research and Innovation (project no.2019062813376603 and 2020052517411298).
Patient blood sampling was performed with written informed consent according to the Declaration of Helsinki and its later amendments.DNA extraction as well as blood/DNA storage and utilization followed institutional IRB-accepted protocols (CE-2022-3) (Medical Faculty, University of Strasbourg).Patients were from France and Spain.