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. Studies with electron microscopes (EMs) have revealed that, in striated muscle cells, the SR is organized into numerous interconnected tubules forming a network of longitudinal elements surrounding each myofibril [1]. These elongated tubules, known as longitudinal SR (l-SR), are dedicated to the removal of Ca²⁺ from the cytosol, an activity operated by the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPases (SERCA) that actively pump Ca²⁺ from the cytoplasm to the lumen of the SR. In skeletal muscle fibers, the l-SR is localized around the A and I bands of each sarcomere. At regular intervals, which correspond to the borders between A and I bands of sarcomeres, the tubules of the l-SR merge into enlarged sac-shaped structures, known as terminal cisternae. Here, two terminal cisternae are positioned at the opposite sides of tubular infoldings in direct continuity with the plasma membrane, named transverse tubules (TT) (Figure 1). The structure formed by two terminal cisternae and one TT, called a "triad", represents the membrane platform where several dedicated proteins operate in transducing the depolarization of the plasma membrane into Ca²⁺ release from the SR, a mechanism known as excitation–contraction coupling (ECC). The region of the terminal cisternae that opposes the TT membrane is called junctional SR (j-SR), and, in this region, the Ryanodine Receptors type 1 (RyR1) Ca²⁺ release channels are localized.

The triad is the membrane structure that supports ECC in skeletal muscle fibers by allowing the interaction between the dihydropyridine receptor (DHPR) and RyR1 [12]. The DHPR is a voltage-dependent L-type Ca²⁺ channel located on the TT that, following sarcolemma depolarization induced by motor-neuron stimulation, undergoes a conformational change that allows the opening of RyR1, the Ca²⁺ channel located on the terminal cisternae of the j-SR, resulting in a massive Ca²⁺ efflux from the SR into the myoplasm and activation of muscle contraction [13].

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 Ca²⁺ binding proteins calsequestrin (CASQ) and histidine-rich calcium (HRC) binding protein ^{[14][23][106][107]}[14,23,106,107] (Figure 3).

The l-SR is mainly involved in Ca²⁺ re-uptake, although some ligand-gated Ca²⁺ channels, such as inositol 1,4,5-trisphosphate receptors (InsP₃R), which release Ca²⁺ to regulate signaling pathways apart from myofibrils contraction, are also present here [198]. SERCA pumps play the most important role in SR Ca²⁺ replenishing. SERCAs belong to the P-type ATPases family, a large superfamily of integral membrane proteins that pump ions and lipids across cellular membranes using the energy derived from ATP hydrolysis. The P-type ATPases family is composed of five subfamilies, defined as P1–P5. SERCAs belong to the P2A-ATPases subfamily and are specific for Ca²⁺ transport, while P4-ATPases act as phospholipid flippases to maintain lipid asymmetry in a variety of membranes ^[199][200][199,200]. SERCA pumps can transport two Ca²⁺ 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][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][204][205]}[203,204,205]. 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][207][208]}[206,207,208]. 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][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][211,212], and differences in Ca²⁺ 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][214][215]}[213,214,215] 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][217][218]}[216,217,218]. 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 Ca²⁺ concentrations, the inhibitory effect of PLN is mitigated due to Ca²⁺/calmodulin kinase (CaMKII)- and/or Protein Kinase A (PKA)-dependent phosphorylation ^{[219][220][221]}[219,220,221]. 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 Ca²⁺, SLN uncouples ATP hydrolysis from Ca²⁺ 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][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 Ca²⁺ binding protein, namely, Sarcalumenin (SAR) [224], which colocalizes and interacts with SERCA2 [225]. Furthermore, SAR has been shown to play an essential role in maintaining cardiac function under biomechanical stresses and endurance exercise training ^[226][227][226,227]. Mutations in SERCA1 are associated with Brody disease, an extremely rare autosomal recessive myopathy (with a prevalence estimated at around 1 in 10 million births) characterized by muscle stiffness, myalgia and muscle cramps, aggravation of symptoms upon exposure to cold temperatures, and, in a smaller percentage of patients, muscle weakness. It is basically characterized by exercise-induced impairment of fast-twitch skeletal muscle relaxation due to diminished SERCA1 activity. This increased levels of cytosolic Ca²⁺ due to SERCA1 impairment directly contribute to muscle stiffness ^{[228][229][230]}[228,229,230].

Although SERCAs represent the key enzymes that contribute to sarcoplasmic Ca²⁺ clearance following muscle contraction, a trans-sarcolemmal flux toward the extracellular environment also occurs in striated muscles. This is mediated by Plasma Membrane Calcium ATPases (PMCAs) and Na⁺/Ca²⁺ exchangers localized in the sarcolemma. Similar to SERCAs, PMCAs also belong to the P-type ATPase superfamily. They can transport one Ca²⁺ for each hydrolyzed ATP and are directly activated by interaction with Ca²⁺-calmodulin, which enhances PMCAs affinity for Ca²⁺ [231]. PMCAs are encoded by four different genes (ATP2B1-4) that, by alternative splicing, generate multiple isoforms [232]. Among these, PMCA3 and PMCA1 were found to be expressed in skeletal muscle, and PMCA1 was specifically localized on the T-tubule membrane ^[233][234][233,234]. NCX catalyzes the exchange of three Na⁺ ions and one Ca²⁺ ion across the sarcolemma in a high capacity and low Ca²⁺ affinity manner. Depending on the electrochemical gradient across the plasma membrane, NCX can either extrude intracellular Ca²⁺ (according to the so-called forward mechanism) or take up extracellular Ca²⁺ (a reverse mechanism) [235]. In mammals, three genes, NCX1, NCX2, and NCX3, are present; NCX3 is expressed in brain and adult skeletal muscle ^[236][237][236,237], while NCX1 is expressed in developing skeletal muscles and in cardiac muscle [237]. The role of NCX has been mainly defined in cardiac muscle cells, where it represents the major mechanism of Ca²⁺ extrusion from the cytoplasm during diastole ^[238][239][238,239].

Entry of Ca²⁺ from the extracellular environment in response to a decrease in the endoplasmic reticulum Ca²⁺ content is a ubiquitous mechanism present in all cell types. The key elements of this pathway, named Store Operated Calcium Entry (SOCE), are STIM1 and Orai1 proteins [240]. STIM1 is a transmembrane protein localized in the ER membrane that acts as a sensor of luminal Ca²⁺ content; when intracellular Ca²⁺ stores are full, it is in a Ca²⁺-bound monomeric conformation. Depletion of the intracellular Ca²⁺ store results in dissociation of Ca²⁺ from STIM1, which forms aggregates that relocate at ER–plasma membrane contact sites, where they interact with and activate the Orai1 channels on the plasma membrane, allowing Ca²⁺ entry from the extracellular environment and refill of intracellular stores. Ca²⁺ entry from the extracellular environment through the SOCE mechanism is also present in skeletal muscle [241]. More recently, the requirement of extracellular Ca²⁺ to refill the SR Ca²⁺ stores, after intense, prolonged activity, has been shown to result in the assembly of additional intracellular junctions between the SR and the plasma membrane, providing additional space for STIM1 and Orai1 interaction. These newly identified sites, named Ca²⁺ Entry Units (CEU), support increased Ca²⁺ entry via Orai1, and their activity appears to contribute to improving fatigue resistance under continuous muscle activity ^{[242][243][244]}[242,243,244] (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][246][247][248][249]}[245,246,247,248,249]. More recently, mutations in CASQ1 have also been identified in TAM patients ^[189][190][189,190].

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][252][253][254]}[251,252,253,254]. 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][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 Ca²⁺ uniporter (MCU), a low-affinity Ca²⁺ transport protein (Figure 4). It has been shown that RyR1 opening results in the formation of local micro-domains, where Ca²⁺ concentration may be up to 10 folds higher than in the rest of the cytoplasm [259]. This high Ca²⁺ concentration allows the transfer of Ca²⁺ to the mitochondrial matrix through the uniporter [260]. From a functional point of view, increases in mitochondrial Ca²⁺ concentration regulate ATP production by enhancing the synthesis of NADH and FADH2 ^[261][262][261,262], thus linking ATP production to muscle demand. Accordingly, slow-twitch fibers in skeletal muscle that are highly oxidative have the highest mitochondrial content compared to fast-twitch fibers; as a result, MCU deletion results in impairment in muscle force and in oxidative metabolism [263]. In cardiac muscle, the MCU-mediated Ca²⁺ influx was shown to play an important role during the fight-or-flight response following catecholaminergic stimulation [264].

Alterations of SR/mitochondria interaction or of mitochondrial morphology and function in striated muscles are often observed in human muscle diseases or mouse models of muscle diseases characterized by alterations in Ca²⁺ signaling ^{[251][265][266][267]}[251,265,266,267]. However, the exact mechanisms linking alterations of MAM, mitochondrial function, and muscle diseases are still to be defined.

This rentryview started with the statement that the SR is a specialized form of ER of muscle cells dedicated to Ca²⁺ handling. One may therefore ask what happened to the ER in striated muscle cells. In eukaryotic cells, the ER is dedicated to Ca²⁺ 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 ^[106][269][106,269]. 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].