ORAI1 and tubular aggregate myopathy

Muscle contraction

Skeletal muscle contraction occurs when an action potential depolarizes the sarcolemma and induces a transient Ca²⁺ elevation, through the so called ECC (Fig. 2B). The gating of DHPR voltage–sensitive Ca²⁺ channels is followed by Ca²⁺ release from RyR on the SR membrane and allows myofilament contraction (Fig 15). Ca²⁺ binding to troponin C mediates the conformational change of the troponin complex (made of troponin C, I, T and tropomyosin) that exposes myosin binding sites on actin thin filaments. The heads of myosin thick filaments

Figure 14. ORAI1’s essential residues. Redrawn from Prakriya and Lewis (2015).

Side view of the four TM domains of an ORAI1 subunit. V102 and R91 (orange) are the two channel gates. E106, D110, D112, and D114 (black) are involved in Ca²⁺ selectivity. Cysteines C126, C142 and C195 (green) are reactive to oxidants. Extracellular (E190) and intracellular (H155) pH sensors are indicated in blue. E106, D110, and D112 are also sensitive to external pH changes. The glycosylation site (N223, pink) is located in the loop III-IV. Residues of the N-terminus that undergo phosphorylation (S27 and S30) are shown in brown. The two STIM1-binding leucines L273 and L276 are located in the TM4 helical extension (yellow). In red are indicated mutations associated with tubular aggregate myopathy (TAM).

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use the energy from ATP hydrolysis to attach and slide along actin filaments. The subsequent shortening of sarcomeres is responsible for muscle fiber contraction. The attachment of new ATP molecules releases myosin heads from thin filaments, and the contraction cycle can resume (Farah and Reinach, 1995). Long–lasting Ca²⁺ elevations are needed to sustain contraction over time, and this is where SOCE comes in. Indeed, the inhibition of SOCE reduces contractile strength and muscle endurance (Thornton et al., 2011). Orai1 deficient zebra fishes display abnormal sarcomeres structure, suffer from skeletal myopathy and die from heart failure (Volkers et al., 2012). While reduced Ca²⁺ levels impair sustained muscle contraction, muscle fiber Ca²⁺ overload is also deleterious. Gain–of–function mutations in STIM1 and ORAI1 genes as well as a mutation impairing CASQ1 polymerization lead to intracellular Ca²⁺

accumulation and were related to TAM (Bohm et al., 2013; Misceo et al., 2014; Nesin et al., 2014; Endo et al., 2015; Bohm et al., 2017; Bohm et al., 2018).

Figure 15. Scheme of Ca²⁺-induced sarcomere shortening.

(A) DHPR and RyR opening induce Ca²⁺ entry and SR Ca²⁺ release respectively (see Fig. 3). Ca²⁺ ions binding to troponin C mediates the conformational change of the troponin (purple)-tropomyosin (red) complex, and exposes myosin binding sites on the actin filaments (green). Myosin (orange) heads use the energy from ATP hydrolysis to attach actin filaments. (B) The motor energy of myosin heads moves actin filaments. (C) Myosin and actin sliding is responsible for sarcomere shortening.

- 39 - Tubular aggregate myopathy (TAM)

TAM is a rare genetic disorder of unknown prevalence, with mainly autosomal dominant transmission. Only occasional cases of recessive inheritance or de novo mutation were described (Hedberg et al., 2014; Bohm et al., 2017). The Ca²⁺ imbalance observed in TAM is associated with myalgia, muscle stiffness, cramps and increased fatigability, all exacerbated by exercise. Asymptomatic cases are very rare. Proximal muscles of the lower limbs are preferentially affected. The clinical features usually appear during childhood and progress with age. Often, young patients present an uncommon tip–toe walking, difficulties to run, to jump or to get up from sitting or squatting positions, joint contractures and delayed motor milestones. However, the disease onset, severity, progression and prognosis is very variable among patients. Electromyogram and muscle MRI sometimes show electrical signs of myopathy and fibro–fatty accumulation in affected muscles. But the diagnosis mainly relies on elevated serum concentrations of creatine kinase (CK) – an indicator of muscle damage – and the presence of sarcoplasmic tubular aggregates in muscle biopsies. Usually, blood Ca²⁺ levels are normal.

Formation of aggregates normally occurs in aging muscles, can be potentiated by alcohol consumption or steroid treatment and is observed in pathological conditions such as TAM, myasthenia or periodic paralysis (Engel, 1966; Chui et al., 1975; Gilchrist et al., 1991; Belaya et al., 2012). In fact, formation of sarcoplasmic tubules was proposed to be a protecting mechanism through which SR buffers the excessive Ca²⁺ ions to prevent muscle fibers hypercontraction and necrosis (Salviati et al., 1985; Chevessier et al., 2004). Aggregates are detected in both types of skeletal muscle fibers: slow twitch (type I) – specialized in long–term contraction and solicited during prolonged efforts of low intensity – and glycolytic or oxidative fast twitch (type II) – involved in more intensive activities. However, they are more commonly observed in type II fibers and are concomitant with type II atrophy, leading to an increased percentage of type I fibers in muscle biopsies (Rosenberg et al., 1985; Bohm et al., 2013). Fiber size variability and nuclei internalization are other histological features of TAM (Fig. 16).

Tubular aggregates appear red in Gomori trichrome stained sections, and dark blue with nicotinamide adenine dinucleotide–tetrazolium reductase (NADH–TR). They are negative with succinic dehydrogenase (SDH), thus incompatible with mitochondrial localization and indicating a reticulum origin. Aggregates are better appreciated by electron micrographs (EM) that show single or double–walled tubules of irregular diameters and length, and are classified in three categories depending on their diameter and density (Table 2). Immunofluorescence

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experiments show aggregation and co–localization of DHPR, RyR1, SERCA, CASQ1 and STIM1, confirming the sarcoplasmic origin of tubular aggregates and suggesting the apposition of sarcolemma around SR tubules (Bohm et al., 2013; Bohm et al., 2014; Bohm et al., 2017).

Figure 16. Histological and ultrastructural aspects of tubular aggregates. From Böhm et al. (2013).

(A) Tubular aggregates appear red with modified Gomori trichrome (left), dark with NADH-TR staining (middle), and are SDH negative (right). Arrows indicate internalized nuclei. (B) Electron micrograph showing single- and double-walled tubular aggregates of variable sizes and diameters (top: transversal section, bottom: longitudinal section). (C) Immunofluorescence showing accumulation of SERCA1, RyR1 and STIM1 in tubular aggregates and their co-localization, indicating that tubular aggregates are of sarcoplasmic origin. In the upper panel, control muscle sections from healthy patients show a homogeneous distribution of SERCA1 and RyR1, and enrichment of STIM1 close to the sarcolemma.

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TAM mainly affects skeletal muscle, but concomitant extramuscular symptoms are often observed. York syndrome combines TAM with mitochondrial myopathy and platelet defects (White and Gunay-Aygun, 2011; White et al., 2013; Markello et al., 2015). The diagnosis of York syndrome is based on the presence of giant opaque inclusions in megakaryocytes and platelets, sometimes accompanied by thrombocytopenia. The combination of TAM with bleeding diathesis, thrombocytopenia and congenital miosis is known as the Stormorken syndrome and can be associated with anemia, asplenia, ichthyosis, headaches, short stature, and cognitive deficiency (Stormorken et al., 1985; Misceo et al., 2014; Morin et al., 2014; Nesin et al., 2014).

The overlap of TAM, York and Stormorken syndromes indicate that they are spectra of the same disease. No mouse model of York syndrome is available yet. A knock–in mouse model carrying the Stormorken STIM1 mutation R304W was recently established and is currently characterized (by the Frengen and Misceo group, Institute of Clinical Medicine, Norway).

However, TAM features were described in male SAMP8 (senescence accelerated, (Nishikawa et al., 2000), male MRL^+/+ (Kuncl et al., 1989), CK deficient (Novotova et al., 2002) and caveolin deficient (Schubert et al., 2007) mice. An ORAI1 gain–of–function mouse model still needs to be developed. These existing models as well as future ORAI1 specific TAM mice will be very valuable tools to assess the efficiency of SOCE inhibitors and to improve therapeutic strategies for patients suffering from TAM.

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