The sarcoplasmic reticulum (SR) is a specialized form of the endoplasmic reticulum of muscle cells, dedicated to calcium ion (Ca2+) handling, necessary for muscle contraction and relaxation.
- muscle
- Ca2+
- myopathy
- intracellular membrane
1. Introduction
Figure 1. Organization of the sarcoplasmic reticulum in skeletal muscle cells. The regular alternation of anisotropic (A) and isotropic (I) bands along a single myofibril is depicted in the upper part of the image. A and I bands are bisected by the M band and the Z disk, respectively. The portion of the myofibril between two Z disks is the sarcomere. The SR is composed of tubules and cisternae surrounding each myofibril. The elongated tubules are known as longitudinal SR (l-SR); they are dedicated to the removal of Ca2+ from the cytosol and are localized around the A and I band of each sarcomere. At the borders between the A and I band, the l-SR merges to form the terminal cisternae. These are positioned at the opposite sides of a transverse tubule (TT); the structure formed by two terminal cisternae and one TT is called a "triad". The region of the terminal cisternae that opposes the TT membrane is called junctional SR (j-SR). Adapted from “Myofibril Structure” by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates, accessed on 14 March 2022.sAnk1.5 and Obscurin Stabilize the SR around the Myofibrils
2. The Triad, a Unique Membrane System of the Skeletal Muscle
Triad Biogenesis, Repair, and Maintenance
Figure 2. In vitro differentiation of primary rat myocytes.Primary rat myocytes induced to differentiate for 1 to 18 days were decorated with antibodies against RyR1 to label the j-SR. At the beginning of differentiation, RyRs show a diffuse distribution in the SR. Starting from day 4 of differentiation, RyRs form clusters, and the SR progressively forms peripheral couplings and/or diads with the TT. Triads form during the following days, and at the end of differentiation, they acquire their transverse orientation and localize at the borders between the A and I bands of the sarcomeres.
Figure 3. Schematic representation of the main proteins accommodated in TT, j-SR, and l-SR. Protein localization and reciprocal interactions are schematized as detailed in the text. Red arrows indicate Ca2+ fluxes (red dots) through RyR1, Orai1, and SERCA pumps. RyR1 opens following interaction with DHPR; Orai1 opens following interaction with STIM1 aggregates, which in turn are induced by a reduction in Ca2+ levels in the SR; SERCA pumps actively transport Ca2+ from the cytosol to l-SR; PLN or SLN act as SERCA inhibitors. DNM2, Cavin-4, BIN1, CAV-3, and MTM1 are involved in the maintenance of TT architecture and stability. They also participate in TT formation (not shown) and, together with DYSF, contribute to vesicle trafficking during the repair of the damaged plasma membrane (see text for additional details). For simplicity, not all proteins and/or protein complexes depicted, including cytoskeleton components, are positioned on both sides of the triad, as it occurs physiologically. The following is a list of acronyms depicted in Figure 3: BIN1 (Bridging integrator-1/Amphiphysin 2); CASQ1 (Calsequestrin 1); CAV-3 (Caveolin-3); CKAP4 (Cytoskeleton-associated protein 4/Climp63); DHPR (dihydropyridine receptor); DNM2 (Dynamin 2); DYSF (Dysferlin); HRC (Histidine-Rich Calcium binding protein); JNT (Junctin); JP45 (J-SR protein 1); JPH1 (Junctophilin 1); j-SR (junctional sarcoplasmic reticulum); l-SR (longitudinal sarcoplasmic reticulum); MG29 (Mitsugumin-29); MG53 (Mitsugumin-53); MTM1 (Myotubularin); PLN (Phospholamban); RyR1 (Type 1 Ryanodine Receptor); SAR (Sarcalumenin); SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase); SLN (Sarcolipin); STIM1 (Stromal Interaction Molecule 1); TRDN (Triadin); TRPC3 (Transient Receptor Potential Cation Channel 3); T-tubule (transverse tubule). Adapted from [23].3. The Protein Complex of the ECC
At triads, several proteins participate in the ECC process, including integral membrane proteins such as RyR1, DHPR, triadin, junctin, j-SR protein 1 (jp45), and mitsugumin-56, the STAC3 adaptor protein, or luminal proteins such as the Ca
3. The Protein Complex of the ECC
2+ binding proteins calsequestrin (CASQ) and histidine-rich calcium (HRC) binding protein [14,105–107] (
Figure 3).
3.1. RyR1, DHPR, and STAC3 Are Essential for ECC
RyRs are a family of Ca
3.1. RyR1, DHPR, and STAC3 Are Essential for ECC
2+
2+
2+
2+
2+
2+ release from intracellular stores [13,108,109]. RyR2 channels are mainly expressed in cardiac muscle, where they are involved in cardiac ECC through a CICR mechanism [105]. RyRs have a tetrameric structure, made by the assembly of four identical monomers, each consisting of a large N-terminal region of about 4300 amino acids extending in the sarcoplasm, while the remaining region contains the six transmembrane helices that anchor each monomer to the SR membrane and contribute to forming the channel pore region, followed by a short cytoplasmic C-terminal tail [110,111]. EM reconstruction studies have shown that RyRs display a mushroom-like form, with the cap in the cytoplasm representing 80% of the volume, and the stalk anchored in the j-SR membrane. In the cytoplasmic region, many domains have been described, referred to as subregions, that represent binding sites for several auxiliary proteins and molecules that contribute to regulating the opening and closing of the channel [110–112]. Calmodulin (CaM) was proposed to bind at different regions of RyR1 channels, acting either as a weak activator, at nanomolar Ca
2+
2+
2+
2+/CaM-dependent protein kinase II (CaMKII), and phosphatases (PP1, PP2A, and PDE4D3) [111,120].
DHPRs are L-type voltage-gated Ca
2+
s
V
s
a
s
s
a
s
2+
2+
2+ channel activity of DHPR and accelerates DHPR activation. Interestingly, this activity is unique to RyR1 since RyR2 was found not to be able to restore either orthograde or retrograde signaling. Different regions in DHPR and RyR1 have been proposed to be important for their reciprocal association and regulation; the II-III loop of the α1
s subunit is generally accepted to be involved in both orthograde and retrograde coupling, while the site of interaction on RyR1 has not yet been completely defined [14,127,129].
STAC3
is a recently identified component of the ECC machinery. It is an adaptor protein that supports the trafficking of the α1s subunit of DHPR and regulates the coupling of DHPR with RyR1 [129–132]. STAC3 knockout mice present severe defects in muscle development, mass, and morphology; they are completely paralyzed and die shortly after birth [132]. Mutations in RYR1, DHPR, and STAC3 were identified in several human congenital myopathies [119,133–136], see
Table 1.
3.2. Triadin and Junctin (JNT), Two Integral Membrane Proteins That Regulate ECC
Triadin is encoded by the TRDN gene that generates, via alternative splicing, four different isoforms named according to their molecular weight: the skeletal muscle isoforms are Trisk 95 and Trisk 51, both displaying a triadic localization; the main cardiac isoform Trisk 32 (also known as CT1) and Trisk 49, which does not localize at the j-SR in skeletal muscle fibers [137,138]. Triadin isoforms share a common cytoplasmic N-terminal and transmembrane domain but differ in the length and composition of their luminal C-terminal segment [139]. Trisk 95 contains two cysteine residues in its luminal domain, C270 and C649, that enable self-multimerization and is targeted at triads by specific domains localized both in the cytoplasmic and the luminal regions [140,141]. Triadin acts as a functional regulator of ECC by interacting with RyRs [142,143], junctin [144,145], calsequestrin [141,146–148], and the histidine-rich Ca
| Causative Gene (S) * | Inheritance | Histological Features |
Clinical Features |
|
|---|---|---|---|---|
| Central Core Disease (CCD) | RyR1 > 90% CACNA1S |
AD or AR |
|
|
| Multiminicore Disease (MMD) | RyR1 CACNA1S |
AR |
|
|
| Centronuclear Myopathy (CNM) | RyR1~12% | AR |
|
|
| Congenital Fibre Type Disproportion (CFTD) | RyR1~20% | AR |
|
|
| Dusty Core Disease (DUCD) | RyR1 | AR |
|
|
| Core Rod Myopathy (CRM) | RyR1 | AD or AR |
|
|
| Malignant Hyperthermia (MH) | RyR1 CACNA1S STAC3 |
AD |
|
|
3.2. Triadin and Junctin (JNT), Two Integral Membrane Proteins That Regulate ECC
2+-binding protein (HRC) [149]. Triadin may also play a structural role in supporting triad architecture by interacting with the microtubule-binding protein Climp-63, also known as Cytoskeleton-associated protein 4 (CKAP4) [150]. Triadin knockout mice present both ECC alterations and abnormal triads [151,152]. In contrast to heart muscle, where mutations in triadin are associated with heart disease, no skeletal muscle disease has been associated with triadin so far.
Junctin is structurally similar to triadin and, like triadin, can bind calsequestrin and the ryanodine receptor [144,145,153–155]. Junctin knockout mice exhibited increased contractility and Ca
2+
2+
3.3. Calsequestrin- and Histidine-Rich Calcium-Binding Protein Store Ca
2+ in the SR Lumen
In skeletal muscle, two isoforms of calsequestrin (CASQ) have been identified: CASQ1 and CASQ2. CASQ1 is expressed in fast- and slow-twitch skeletal muscle fibers, whereas CASQ2 is expressed in slow-twitch skeletal muscle fibers and in cardiac muscle [160]. The two isoforms present a high sequence homology and basically only differ in their acidic C-terminus [161]. CASQs are intra-luminal SR soluble proteins with high capacity and low-affinity Ca
in the SR Lumen
2+
2+
2+
2+
2+ concentration and the presence of CASQ-binding proteins or post-translational modification [168–171]. During sustained SR Ca
2+
2+release aimed to prevent dangerous levels of SR Ca
2+ depletion. Accordingly, it has been proposed that CASQ1 depolymerization may represent the intracellular switch that induces RyR1 closing [172].
In skeletal muscle, CASQs interact with RyR1, triadin, and junctin, forming a quaternary Ca
2+ release complex [144,145,148,173,174]. However, how these interactions regulate ECC still remains to be defined, since different studies report that CASQs may either inhibit or activate or even have no effect on the opening of RyRs [169,174–181]. CASQ1 knockout mice show mild atrophy, narrower terminal cisternae, and proliferation of multilayered junctions, as well as mitochondria alterations. They exhibit significantly reduced SR Ca
2+
2+
2+
2+ content [186,187]. Mutations in the CASQ1 gene have been identified in patients affected by a rare vacuolar myopathy [188] and in patients with tubular Aggregate Myopathy, TAM [189,190].
HRC is far less abundant than CASQs, suggesting that it works as a secondary calcium-binding protein [191,192]. It is composed of a conserved N-terminal domain, a C-terminal cysteine-rich region, and a central histidine-rich region, which allow HRC to bind Ca
2+
2+
2+
2+concentration, the protein shifts to a monomeric form [195]. Although considered as a secondary calcium-binding protein, HRC appears to play a non-secondary role in regulating Ca
2+
4. SERCA Pumps at the l-SR Are Responsible for Ca
2+ Re-Uptake from the Sarcoplasm
The l-SR is mainly involved in Ca
Re-Uptake from the Sarcoplasm
2+
2+
3
2+
2+
2+
2+ ions for each hydrolyzed ATP. In vertebrates, SERCA pumps are encoded by three different genes, named ATP2A1-3. More than 10 protein variants are generated through alternative splicing occurring in the 3′-end of the main transcripts [201,202].
ATP2A1 encodes two major skeletal muscle proteins: SERCA1a and SERCA1b, highly expressed in fast-twitch skeletal muscle fibers. SERCA1b is expressed during neonatal stages and in regenerating muscles, while it is replaced by SERCA1a in adult skeletal muscles [203–205]. Four isoforms of SERCA2 (a–d) are generated by alternative splicing of the ATP2A2 gene. SERCA 2a is expressed in cardiac muscle, slow-twitch skeletal muscle fibers, and smooth muscle cells. SERCA2b is reported to be ubiquitously expressed, and it represents the main SERCA isoform in the brain [206–208]. mRNA of SERCA2c was found in epithelial, mesenchymal, and hematopoietic cell lines, as well as in primary human monocytes [206] and cardiac muscle, while SERCA 2d has been identified in skeletal muscle, although its role has not yet been fully elucidated [209]. ATP2A3 codes for six isoforms (SERCA3a-f), mostly identified in non-muscle cells, although SERCA3a, 3d, and 3f were also detected in cardiac muscle cells [209,210]. The primary structure of the different SERCA isoforms is highly conserved. Nevertheless, the SERCA variants differ in their enzymatic properties. For instance, SERCA1a displays a maximal activity that is two-fold higher than that of SERCA2a [211,212], and differences in Ca
2+ affinities have also been reported between SERCA2b and SERCA3 [212]. SERCA activity is finely regulated by numerous small proteins. The first identified regulator of SERCA is Phospholamban (PLN), which exerts an inhibitory effect on SERCA in cardiac muscle [213–215] and, to a lesser extent, in smooth muscle and slow-twitch oxidative skeletal muscle fibers where, however, its functional role appears to be marginal [216–218]. PLN exists in two forms, monomeric and pentameric. The first is considered the “active” inhibitory form, while the pentameric state is considered the “inactive” storage form of PLN. PLN regulation occurs through one-to-one interaction with SERCA [218]. At high cytosolic Ca
2+
2+/calmodulin kinase (CaMKII)- and/or Protein Kinase A (PKA)-dependent phosphorylation [219–221]. A second inhibitor of SERCA in skeletal muscle and atria is represented by sarcolipin (SLN). In contrast to PLN, which inhibits SERCA activity by reducing its affinity for Ca
2+
2+transport [222], thus promoting futile cycling, resulting in ATP consumption and increased thermogenesis. Indeed, SLN knockout mice do not show any visible phenotype but show compromised thermogenic capacity when exposed to cold [222,223]. This suggests that the role of SLN may be related to muscle functions linked to body metabolism and heat production rather than contraction [223]. Similar to the j-SR, the l-SR also contains a Ca
2+
2+ due to SERCA1 impairment directly contribute to muscle stiffness [228–230].
Although SERCAs represent the key enzymes that contribute to sarcoplasmic Ca
2+
+
2+
2+
2+
2+
+
2+
2+
2+
2+
2+
5. Ca
2+
Entry Units (CEU): Novel SR/Plasma Membrane Contact Sites to Refill Intracellular Ca
2+ Stores
Entry of Ca
Stores
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+ entry via Orai1, and their activity appears to contribute to improving fatigue resistance under continuous muscle activity [242–244] (Figure 4). Mutations in Orai1 and STIM1 have been identified in patients with tubular aggregate myopathy (TAM), a rare genetic disease characterized by accumulation, in type-2 fibers, of highly ordered membrane tubules containing SR proteins, such as CASQ1, SERCA, triadin, RyR1, and STIM1 [245–249]. More recently, mutations in CASQ1 have also been identified in TAM patients [189,190].
Figure 4. Schematic representation of membrane contact sites contributed by the SR. In addition to triads, the SR contributes to the formation of additional membrane contact sites in muscle cells. Depletion of intracellular Ca
Figure 4. Schematic representation of membrane contact sites contributed by the SR. In addition to triads, the SR contributes to the formation of additional membrane contact sites in muscle cells. Depletion of intracellular Ca2+
2+
2+
2+
2+
2+storage system between the SR and the nuclear envelope. The inner nuclear membrane forms invaginations that enter the nuclear matrix to support intra-nuclear Ca
2+ signaling. Created with BioRender.com. accessed on 14 march 2022
6. Mitochondria-Associated Membranes (MAM)
Interaction between the ER/SR and mitochondria occurs at specific membrane contact sites, generally defined as Mitochondria Associated Membranes (MAMs), populated by several proteins and protein complexes that act as tethers between the SR and the outer mitochondrial membrane [250]. In striated muscles, mitochondria are mostly positioned in the inter-myofibrillar spaces adjacent to triads, although contacts between mitochondria and the longitudinal SR have also been observed [251–254]. Association with the SR is mediated by the voltage-dependent anion channel 1 (VDAC) [255]. In cardiac muscle, it has been proposed that VDAC interacts with RyR2 [256]. Mitofusin-2 (MFN-2) also contributes to tethering SR and mitochondria in skeletal muscles [257,258]. Interestingly, during mouse skeletal muscle development, mitochondria redistribute from a longitudinal arrangement towards a triad-associated distribution, with an increase in the frequency of contacts until the first four months after birth [258]. These contacts are aligned with domains of the inner mitochondrial membrane enriched in the mitochondrial Ca
6. Mitochondria-Associated Membranes (MAM)
2+
2+
Figure 4
2+
2+
2+
2+
2+
2+ signaling [251,265–267]. However, the exact mechanisms linking alterations of MAM, mitochondrial function, and muscle diseases are still to be defined.
7. The SR and the ER: Two Faces of a Single Organelle
This review started with the statement that the SR is a specialized form of ER of muscle cells dedicated to Ca
7. The SR and the ER: Two Faces of a Single Organelle
2+
2+ storage, protein synthesis and folding, and lipid and sterol synthesis. Although ER-related functions are clearly present in striated muscles, the distribution of the ER within the SR membranes is less evident. The recognized idea is that the SR and the ER represent a continuous membrane system made of different specialized subdomains [268]. Compartmentalization of ER and SR markers was demonstrated in skeletal muscle fibers, where ER-specific proteins were detected at the perinuclear region and in two distinct rough ER sub-compartments: the first, in correspondence of the I band that does not apparently contain ER exit sites, and the second, near the Z disk, which sustains export activity towards the Golgi [105,269]. Similarly, in cardiac muscle cells, protein synthesis was found to occur not only around the nuclei but also within ER/SR membranes surrounding the sarcomeres; interestingly, translation of some SR membrane proteins has been proposed to occur starting from pools of mRNAs that are transported from the perinuclear region towards ER/SR protein-synthesis sites through a microtubule-dependent system [270]. A less-clear distinction can be observed in smooth muscle, where ER and SR markers are basically overlapping [271].
7.1. Add
Additional Contact Sites Contributed by the ER/SR
In eukaryotic cells, the ER is engaged in a complex network of interactions with many intracellular components, such as the nuclear envelope (NE), the plasma membrane, mitochondria, the Golgi complex, peroxisomes, endosomes, lysosomes, and lipid droplets [272]. All these interactions have been less characterized in striated muscle cells. Lysosome–SR nanojunctions were described in pulmonary arterial myocytes (Figure 4). These junctions were identified between clusters of lysosomes and perinuclear regions of the SR rich in RyR3 and are believed to provide an intracellular structure involved in the regulation of specific Ca
2+
2+
2+
2+
2+
2+
2+
2+signaling (Figure 4). The NE is formed by two phospholipid bilayers that form the inner nuclear membrane and the outer nuclear membrane, outlining an internal space called the perinuclear cisternae. The outer nuclear membrane is continuous with the membrane of the rough ER; this connection results in the formation of common luminal space with the ER, which, in muscle cells, allows the arrangement of a continuous Ca
2+
2+signaling [277]. InsP
3
2+release is apparently mainly regulated by RyRs [278,279], while both InsP
3
2+ signaling may be more complex than previously expected [281].
An additional issue is represented by the SR/nuclei juxtaposition within the overall skeletal muscle architecture. In skeletal muscle, nuclei are positioned at the fiber periphery between myofibrils and the sarcolemma, in proximity to T-tubules. The positioning of nuclei is regulated by different mechanisms, including the formation of a specific microtubule network, and is critical for muscle function; indeed, improper myonuclear localization is observed in muscle diseases, such as centronuclear myopathy and muscular dystrophies [282]. The molecular link supporting nuclear juxtaposition with the SR is currently not known. A recent work performed in skeletal muscles of Drosophila melanogaster suggests that this interaction may be mediated by a three-partner connection where amphiphysin 2 on TT binds, at the same time, Ma2/d, a protein present on both TT and SR membranes, and, at the nuclear membrane, Msp30, a protein previously described to mediate connections between the NE and the Z disks [283–285].
8. Concluding Remarks
This review provides an updated view of the structural, molecular, and functional organization of the SR in skeletal muscle, highlighting some of the specialized domains whereby this organelle supports activation of muscle contraction. The historical knowledge that the terminal cisternae of the SR engage with the T-tubules in forming the triad, which represents the first identified membrane contact site, is now expanded by evidence that in skeletal muscle, the SR participates in the assembly of at least two more types of membrane-contact sites.
In one case, the identification of CEUs, dynamic membrane contact sites consisting of stacks of SR cisternae and the extension of T tubules, structurally and functionally different from triads, provides evidence of how skeletal muscle fibers can sustain refilling of intracellular Ca2+ stores under conditions of prolonged activity. The second contact site, MAM, between the SR and mitochondria, has the functional role of synchronizing the mitochondrial energy production rate with the metabolic demand of muscle fibers. Altogether, these new findings are changing the traditional view of the SR as a static organelle by depicting an unexpected dynamic ability to extend its complex structure to support muscle function.
At the same time, there are several points that remain to be clarified. A major open question relates to the precise positioning of triads at the boundary of the I and A bands of the sarcomere. This highly precise pattern, evident in EM micrographs for about a half-century, is waiting for the identification of the proteins that tether triads to the sarcomere with such a regularly repetitive precision. Concerning CEU, we need to identify the molecules that support the dynamic assembly and disassembly of CEU and how Orai1 and STIM1 are recruited at these sites. Mitochondria, like many other organelles, in skeletal muscle, are localized with a well-defined pattern with respect to triads and sarcolemma, a pattern that differs in slow- and fast-twitch fibers. Additionally, in this case, the molecules that anchor mitochondria to these domains of muscle fibers remain to be identified.
In conclusion, we can anticipate that the above list is, certainly, still missing additional SR domains that are yet to be identified; these might probably involve activities related to the regulation of Ca2+ homeostasis, but more likely, additional functions necessary to keep skeletal muscle fibers healthy and fully functional.