For the past three decades, physiologists, biochemists, and structural biologists have examined the roles of the skeletal muscle type of CASQ (CASQ1) in skeletal muscle and revealed that CASQ1 has various important functions as (1) a major Ca2+-buffering protein to maintain the SR with a suitable amount of Ca2+ at each moment, (2) a dynamic Ca2+ sensor in the SR that regulates Ca2+ release from the SR to the cytosol, (3) a structural regulator for the proper formation of terminal cisternae, (4) a reverse-directional regulator of extracellular Ca2+ entries, and (5) a cause of human skeletal muscle diseases. This review is focused on understanding these functions of CASQ1 in the physiological or pathophysiological status of skeletal muscle.Calsequestrin (CASQ) was discovered in rabbit skeletal muscle tissues in 1971 and has been considered simply a passive Ca Although previously considered merely a passive regulator of calcium levels, the protein calsequestrin is now known to perform a range of physiological activities essential to skeletal muscle function. The process of muscle contraction depends on the release of calcium ions from an intracellular structure called the sarcoplasmic reticulum (SR). Calsequestrin was originally identified as a “buffering” factor that maintains adequate calcium reserves in the SR, but Eun Hui Lee and colleagues at the Catholic University of Korea review diverse functions that have since been ascribed to this protein. For example, calsequestrin also helps reinforce the structure of the SR, and actively regulates the flux of calcium ions into muscle cells. Perturbations in calsequestrin function also appear to contribute to a number of muscular disorders, including a potential link to Duchenne muscular dystrophy. To sustain body postures and perform body movements, skeletal muscle contracts or relaxes, and the main key elements governing skeletal muscle contraction and relaxation are well understood2. During contraction and relaxation, the movements of contractile proteins in skeletal muscle cells  are mainly dependent on the cytosolic level of Ca2+ ions that are released from the SR. Dihydropyridine receptor  on the transverse (t)-tubule membrane) senses the depolarization of the t-tubule membrane  in response to acetylcholine that is released from motor neurons. T-tubules are invaginations of the plasma membrane and have a transverse orientation with respect to the main axis of myofibrils. The conformational changes in DHPR due to the action potential subsequently activate ryanodine receptor type 1  via physical interactions . The interaction allows the release of Ca2+ from the SR to the cytosol through RyR1. The released Ca2+ from the SR turns on contractile proteins and evokes skeletal muscle contraction. In short, a transient elevation in cytosolic Ca2+ levels due to Ca2+ release from the SR couples the action potential and muscle contraction ” coupling). EC coupling is defined as an “orthograde signal” from DHPR in the t-tubule membrane to RyR1 in the SR membrane . Three different genes on chromosomes 19, 1, and 15 encode three distinct isoforms of RyRs . RyR1 is predominant in skeletal muscle, whereas RyR3 is expressed at much lower amounts than RyR1 in skeletal muscle. In addition to the physical interaction of DHPR and RyR1, the Ca2+ that is released from SR binds and activates nearby RyR1, which does not interact physically with DHPR. This is called Ca2+-induced Ca2+ release, which contributes to rapid intracellular Ca2+ release from the SR and to the amplification of Ca2+-mediated signals3.Generally, the Ca2+] at rest (nM range) rises to the μM range during skeletal muscle contraction4. To establish the Ca2+ increase, in addition to the Ca2+ from the SR, extracellular Ca2+ entry via Ca2+ channels in the t-tubule membrane, such as Orai1 or transient receptor potential canonical type (TRPC), also participates in the cytosolic Ca2+ increase, especially for skeletal muscle contraction during tetanic stimulation or fatigue7. The relaxation of skeletal muscle involves the reuptake of Ca2+ from the cytosol to the SR to reset the resting cytosolic Ca2+ level and replenish SR Ca2+. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase  is mainly responsible for Ca2+ reuptake, and SERCA1a is the major isoform in adult skeletal muscle8. In addition to SERCA, the Na+/Ca2+ exchanger (NCX) in the plasma membrane, mNCX in the mitochondrial membrane or Ca2+-ATPase in the plasma membrane  also participate in removing Ca2+ from the cytosol under certain conditions, such as a Ca2+ increase involving store-operated Ca2+ entry (SOCE)11. The efficient arrangement of EC coupling-mediating proteins mentioned above along with their regulatory proteins in the triad junction  is crucial for their proper functions and intermolecular interactions14. Therefore, for the successful cycling of the rise and fall in cytosolic Ca2+ level during repetitive skeletal muscle contraction and relaxation during a lifetime, the spatiotemporal distributions of intracellular Ca2+ ions, timely expression of EC coupling-mediating proteins with their regulatory proteins at different levels, and formation of triad junctions should be coordinately prepared with pinpoint accuracy.Cytosolic  in the SR 69. The polymerization of CASQ1 in skinned fibers from rat skeletal muscle theoretically increases the total [Ca2+]SR up to 20 mM, and simultaneously, it maintains a free [Ca2+]SR at approximately 1 mM70, which is important for easier and more efficient Ca2+ reuptake into the SR by SERCA1a during relaxation and for the storage of high [Ca2+] in the SR without harmful osmotic effects. For example, frog skeletal muscle fibers with CASQ1 bear 23 times more Ca2+ than those without CASQ171.The first role of CASQ1 in skeletal muscle is to buffer Ca2+ sensor in the SR. CASQ1 senses [Ca2+] in the SR and modulates Ca2+ release from the SR to the cytosol via RyR1 in a conformation -dependent manner . Under this condition, CASQ1 is depolymerized and no longer inhibits RyR1 activity. CASQ1 also protects the SR from deep Ca2+ depletion, which causes massive migration of CASQ1 away from the JSR membrane and remodeling of the SR75. Under long-lasting membrane depolarization, such as high-frequency stimuli or early stages of muscle fatigue, Ca2+ in the SR is not fully but partly depleted due to the inactivation of RyR1 at a certain moment by limiting the further depolymerization of CASQ1, which prevents excess Ca2+ release from the SR and catastrophic changes in the SR structure. Therefore, it can also be said that by conformational changes (polymerization or depolymerization) in a Ca2+-dependent manner, CASQ1 senses the degree of Ca2+ depletion from the SR and regulates RyR1 activity. In addition, the regulation of RyR1 activity by CASQ1 requires interactions with other proteins (discussed in ‘Regulation of RyR1 activity by a quaternary complex of CASQ1/RyR1/triadin/junctin’ in this review).The second role of CASQ1 in skeletal muscle is as a Ca53. The CASQ1 monomer has three almost identical domains (I–III), each with a topology similar to that of thioredoxin from Escherichia coli . Each domain forms a disk-like shape with a fold of α/β structures (a five-stranded β-sheet in the core flanked by four α-helices (two on each side of the β-sheets). The thioredoxin-like fold occurs at ~10 μM [Ca2+]. The roles of the thioredoxin-like domain in CASQ1 have not been studied. Considering that the thioredoxin system participates in the regulation of metabolism, the antioxidant system, cell signaling pathways, and thioredoxin-dependent chaperone actions76, the presence of the three thioredoxin-like domains in CASQ1 suggests that CASQ1 could be directly or indirectly involved in the thioredoxin system.The 3D structure of CASQ1 was determined at a resolution of 2.4 Å2+]SR78. At 10 μM [Ca2+]SR, CASQ1 exists as a monomer and dissociates from the JSR membrane, which eliminates the inhibitory effect of CASQ1 on RyR1. Two types of dimers facilitate the polymerization of CASQ1. The first type of dimer occurs via a back-to-back interaction. Salt bridges between Glu215-Lys86, Glu215-Lys24, and Glu169-Lys85 from domains I and II stabilize the back-to-back interaction. Two salt bridges between Lys85-Glu223 and between Lys86-Glu215 mediate an interaction between the dihydrobasic sequence  and a binding site for trifluroperazine (an antipsychotic drug), which also contributes to the back-to-back interaction. In the back-to-back interaction, the C-terminal asp-rich/CAS region from each monomer forms a negatively charged pocket within the dimer, and the negative pocket represents a major Ca2+-binding motif in CASQ180. At low [Ca2+], the asp-rich/CAS region can accommodate 6–8 Ca2+ ions, which neutralizes the acidic property of the asp-rich/CAS region and allows back-to-back stacking. The remaining regions of CASQ1 contain additional Ca2+-binding sites that can progressively support more Ca2+-binding as [Ca2+] increases80. The back-to-back interaction during the polymerization of CASQ1 occurs at 10 μM to 1 mM [Ca2+]SR.CASQ1 can exist either as monomers or homopolymers (a wide range of high molecular masses), and polymeric CASQ1 allows various binding to other proteins via the same region, such as the asp-rich/CAS (consecutive aspartate stretch) region. The polymerization of CASQ1 depends on [Ca2+] is further increased. The front-to-front interaction occurs with the insertion of the N-terminal part of one monomer of the dimer into a hydrophobic cleft in the adjacent monomer of another dimer. This process again generates a negatively charged pocket that supports Ca2+-binding. A salt bridge between Glu55–Lys49 contributes to the front-to-front interaction and helps to stabilize CASQ1 polymers. Further polymerization of the CASQ1 tetramers finally forms thin strands  that generate more negative pockets, which confer a higher Ca2+-buffering capacity and the ability to bury hydrophobic side chains of amino acids  on CASQ1. The highly extended structure of polymeric CASQ1sbecomes more compact by the binding of more Ca2+81. Polymeric CASQ1 is stably anchored to the JSR membrane by binding to other proteins  at ~1 mM [Ca2+]SR and can maximally inhibit RyR1 activity50. The polymerization of CASQ1 is inhibited by increasing [K+]. From these reports, both terms of Ca2+-dependent polymerization of CASQ1 or polymerization-dependent Ca2+-binding of CASQ1 are correct. The Ca2+-buffering and Ca2+-sensing abilities of CASQ1 are not two different roles but are functionally and structurally correlated to each other. In short, the Ca2+-dependent dynamic polymerization and depolymerization of CASQ1 provide the SR with a rapidly releasable Ca2+ reservoir during skeletal muscle contraction and a highly negatively charged surface that easily absorbs Ca2+ at rest or during relaxation. The differences in their abilities in front-to-front and back-to-back interactions and in the composition of the asp-rich/CAS region in CASQ1 and CASQ2 generate isoform-specific Ca2+-binding capacities and different Ca2+-dependent polymerization properties82.The second type of dimer occurs via a front-to-front interaction. Tetramerization of CASQ1 occurs via a front-to-front interaction between two dimers (via the N-terminus) that are formed by the back-to-back interaction between two monomers (via the C-terminus) when  at the release site of the SR decreases, which causes the depolymerization of CASQ1 at the site and subsequent release of Ca2+ from the depolymerized CASQ1 to the SR lumen via the loss of Ca2+-binding ability (unbuffering of Ca2+ by CASQ1). More Ca2+ release from the SR to the cytosol can occur by the unbuffering of Ca2+ by depolymerized CASQ1. A further cycle of depolymerization and Ca2+ unbuffering of CASQ1 results in more rapid decay of [Ca2+]SR compared with the beginning of Ca2+ release . Concurrently, the depolymerized CASQ1 detaches from the JSR membrane, which induces the loss of inhibitory action of CASQ1 on RyR1 activity87. Further depolymerization of CASQ1 finally terminates Ca2+ release from the SR through RyR1  when Ca2+ depletion from the SR and the detachment of CASQ1 from the JSR membrane progress further75. In addition, the binding of CASQ1 to RyR1 is also eliminated by increasing [Ca2+]SR to ≥4 mM (probably mimicking the state of the SR at rest), which consequently eliminates the inhibitory effect of CASQ1 on RyR1 activity88.Ca2+ in the area near RyR191. The same luminal KEKE motif of triadin also interacts with the C-terminal loop of RyR193. Triadin physically links CASQ1 to RyR1, forming a bridge between CASQ1 and RyR189. It has also been reported that CASQ1 directly interacts with RyR194. The acidic asp‐rich/CAS region of CASQ1 also interacts with junctin via the KEKE motif96. All these reports suggest that CASQ1 can participate in generating a protein complex with RyR1, junctin and triadin .The acidic asp‐rich/CAS region of CASQ1 in the C-terminus directly interacts with triadin via a KEKE motif in triadin , which helps CASQ1 anchor to the JSR membrane and allows CASQ1 to bind to Ca53. However, according to EM observations of skeletal muscle and other studies on the binding site of CASQ1 to triadin, as mentioned above97, the CASQ1 polymer can form a complex with RyR/triadin/junctin. At 1 mM [Ca2+]SR, the CASQ polymer is stable and anchors to the JSR membrane through triadin and junctin, and further increasing [Ca2+]SR to over 10 mM results in the dissociation of CASQ from triadin and junctin98, which is in accordance with the observation that the CASQ1 polymer at ≥4 mM [Ca2+]SR causes a gradual dissociation of CASQ1 from junctin, triadin and RyR188. Interestingly, both the formation of the quaternary complex and the formation of CASQ1 polymers contribute to the retention of CASQ1 inside the SR in skeletal muscle99.The possibility has been suggested that the N-terminal residues of CASQ1 may also be responsible for its binding to triadin and junctin, and the CASQ1 polymer may not bind to these proteins2+ sensor to modulate the activity of RyR1. In early studies, the functional relevance of the interaction between CASQ1 and RyR1 was controversial. CASQ1 was originally described as an activator of RyR1 in isolated heavy SR vesicles100. In a single-channel study with a planar lipid bilayer, the addition of purified CASQ1 to the luminal side enhanced the activity of purified RyR1101. Overexpression of CASQ1 in C2C12 skeletal myotubes enhanced caffeine- and membrane depolarization-induced intracellular Ca2+ release through RyR1102. Overexpression of a deletion mutant of CASQ1 lacking the C-terminal aspartate-rich/CAS region (the triadin- or junctin-binding domain) in C2C12 myotubes resulted in reduced caffeine- and membrane depolarization-induced Ca2+ release102. Even a case of no functional relationship between CASQ1 and RyR1 was reported 103. Other groups reported that CASQ1 alone could activate RyR1 activity104, but CASQ1 inhibited RyR1 activity by binding to triadin104. Deletion of the C-terminal acidic asp‐rich/CAS region of CASQ1 abolished the inhibitory action of CASQ1 on RyR196. Specifically, in a single-channel study with a planar lipid bilayer, the addition of purified triadin or junctin to the luminal side increased the activity of purified RyR1, and the addition of purified CASQ1 abolished the effect of junctin on RyR1 but not the effect of triadin105. The different compositions of RyR or CASQ isoforms, the different degrees of expression levels of RyR1 or CASQ1 (or the ratio between them), and the different salt concentrations in the various experimental conditions could cause discrepancies in the role of CASQ1 in RyR1 activity. For example, CASQ1 was dissociated from the JSR membrane by increasing the luminal ionic strength , and the inhibitory effect of CASQ1 on RyR1 activity was removed by the dissociation of CASQ167. After all of these reports, it has finally been accepted that at low [Ca2+]SR, CASQ1 functions as a Ca2+ sensor that inhibits RyR1 activity in the presence of triadin at least  to preserve a certain level of Ca2+ in the SR94. Consistently, triadin knockout mice show skeletal muscle weakness with reduced expression of CASQ1106, and a human patient with a triadin mutation also shows skeletal muscle weakness107. For more details on RyR1 activity along with its partners or regulatory proteins under various pathological conditions, including MH, skeletal muscle myopathies, cardiac arrhythmias, epilepsy, neurodegeneration, and pain, please refer to a review article108.CASQ1, triadin, and junctin are not essential for survival or skeletal muscle contraction; however, they are related to RyR1 activity and the gain of EC coupling in skeletal muscle. As mentioned in the previous section of this review, CASQ1 acts as a Ca2+]SR, the phosphorylation status of CASQ1 (see the next section in this review), the polymerization status of CASQ1, binding to other proteins such as triadin and junctin , and even RyR1 activity itself .Overall, the regulation of RyR1 activity by CASQ1 is not a simple one-way process but a very complicated and bidirectional process that is optimized for the fine-tuning of skeletal muscle contraction or relaxation, with dependencies on [Ca2+-binding capacity of CASQ1 by regulating the interaction of CASQ1 with other proteins. CKII (which can phosphorylate serine or threonine residues) is present in the SR lumen in rabbit fast-twitch skeletal muscle109. The threonine located at the beginning of the C-terminus of CASQ1 is the only site of CASQ1 phosphorylated by CKII 55. The acidic region partly overlaps with the triadin-binding region (residues 354-36790) and lies near the electronegative pocket formed by the front-to-front interaction during CASQ1 polymerization53. Phosphorylation of CASQ1 at the threonine residue increases the Ca2+-binding capacity of CASQ1 nearly 2-fold110.The phosphorylation status of CASQ1 also affects the Ca111 and dephosphorylated forms112. It is controversial whether the phosphorylation of CASQ1 affects the interactions between CASQ1 and RyR1 or the inhibitory effect of CASQ1 on RyR1. Only dephosphorylated CASQ1 can enhance RyR1 activity without binding to triadin and junctin (approximately fivefold increase in open probability and approximately twofold increase in mean open time at 1 mM of [Ca2+]SR), and only 1% dephosphorylated CASQ1 in the total CASQ1 is sufficient to enhance RyR1 activity113. However, the affinity of phosphorylated CASQ1 to RyR1 is similar to that of dephosphorylated CASQ194. In contrast, both phosphorylated and dephosphorylated CASQ1 have an inhibitory effect on RyR1 activity at 1 mM [Ca2+]SR and only when the quaternary complex of CASQ1/RyR1/triadin/junctin is intact110. The enhancement of RyR1 activity by dephosphorylated CASQ1 is observed only when CASQ1 is bound to triadin alone110. Considering that functional RyR1 is a very large ion channel , the ambiguity in the relationship between RyR1 activity and CASQ1 could possibly be due to the participation of other proteins that can bind RyR1 and/or exert regulatory effects on RyR1 activity. In addition, interestingly, CASQ1 itself has kinase activity in the presence of Mg-ATP (which is present in the SR) and can autophosphorylate114, suggesting that CASQ1 phosphorylates other proteins in the SR, such as EC coupling-mediating proteins, including RyR1. These various reports surely suggest that the regulation of RyR1 activity by CASQ1 depends on the phosphorylation state of CASQ1, [Ca2+]SR, and the binding of triadin and junctin to CASQ1. Overall, the relationship between the phosphorylation status of CASQ1 and the regulation of RyR1 activity by CASQ1 via interaction between RyR1 and CASQ1 should be further investigated.Isolated CASQ1 exists in both phosphorylated2+-binding capacity of dephosphorylated CASQ1 is reduced by more than one-third compared with that of phosphorylated CASQ1110. Phosphorylation of CASQ1 does not alter CASQ1 polymerization110. Therefore, it is likely that phosphorylated CASQ1 corresponds to polymerized CASQ1.The Ca115. Skeletal muscle tissues and fibers from CASQ1 knockout mice show remodeling of the triad junction, such as a narrow lumen of the JSR caused by shrinking, the proliferation of triad-like multilayered junctions , an increase in the number and volume of mitochondria, and an increase in the number of RyR1 molecules (even in multilayered junctions)  Fig. 115,116. 2+ in the SR of fast-twitch EDL skeletal muscle fibers from CASQ1 knockout mice is reduced compared with that in wild-type fibers115. The appearance of multilayered junctions with RyR1s could be a compensatory response to the reduced SR Ca2+ storage with the shrinkage of terminal cisternae. There is no compensatory increase in the expression of CASQ2 in either fast- or slow-twitch skeletal muscles115. Fast-twitch EDL skeletal muscle fibers from CASQ1 knockout mice display similar free [Ca2+]cytosol but significantly reduced Ca2+ release from the SR in response to electrical stimulation or caffeine compared with wild-type fibers, while slow-twitch soleus skeletal muscle fibers are not affected117. However, tension developed during isometric tetanus for a short duration is not significantly different in the two types of muscle fibers, and the sensitivity to caffeine is also unchanged 116. Fast-twitch EDL skeletal muscle fibers from CASQ1 knockout show prolonged twitch time parameters 115, which conflicts with the reduced Ca2+ release from the SR in response to electrical stimulation or caffeine in the same EDL fibers. These unexpected results can be induced by an impaired ability of Ca2+ reuptake to the SR or a loss of CASQ1-mediated inhibitory effect on RyR1 activity during EC coupling. The increased number of mitochondria is related to the higher resistance of CASQ1 knockout mice to fatigue than wild-type mice115. EDL muscles from CASQ1 knockout mice also show mitochondrial damage with oxidative stress118.The amount of Ca119. Fast-twitch FDB skeletal muscle fibers from single- or double-knockout mice do not sustain prolonged muscle activity  due to faster SR Ca2+ depletion, a decrease in [Ca2+]SR, impaired Ca2+ reuptake to the SR and finally an inability to sustain [Ca2+]cytosol during contraction119. The proper function of fast-twitch FDB skeletal muscle fibers from double-knockout mice requires external Ca2+ entry 119, suggesting a possible relationship between CASQ1 and SOCE (see the next part of this review). Unlike CASQ1 single-knockout mice, CASQ1/CASQ2 double-knockout mice show a more severely damaged structure of the soleus muscle , although both single- and double-knockout mice exhibit significant reductions in body weight and grip strength116. However, under tetanic stimulation (2 s and 100 Hz), the soleus was able to sustain contraction, while active tension in the EDL declined by 70–80%. This discordance in the structural damage and function in the slow-twitch soleus skeletal muscle fibers is explained by the nature of the skeletal muscle type: fast-twitch EDL skeletal muscle fibers need the release of large amounts of Ca2+ from the SR during tetanic stimulation; in contrast, slow-twitch soleus fibers require less Ca2+ and have slower cycling than fast-twitch EDL skeletal muscle fibers.There are no marked differences between mice carrying CASQ1 single- and CASQ1/CASQ2 double-knockouts2+ content due to the reduced expression and lower activity of SERCA along with a reduction in the expression of RyR1 compared to wild-type myotubes101.Transient knockdown of CASQ2 alone or both CASQ1 and CASQ2 in C2C12 myotubes induces a reduction in the SR Ca2+ gradient between the cytosol (approximately 100 nM) and extracellular space (mM range) is maintained , which suggests that extracellular Ca2+ is an effective source of intracellular Ca2+ signals120. SOCE is one extracellular Ca2+ entry route in various cells121. Generally, SOCE is operated by coordinated interactions between STIMs  and Ca2+ release-activated Ca2+ channel proteins . STIM1 and Orai1 are the major isoforms mediating SOCE. STIM1 in the ER membrane senses the depletion of Ca2+ from the ER via its EF-hand motif, and it activates Orai1 in the plasma membrane via a physical interaction between the cytoplasmic C-terminus of STIM1 and the STIM-Orai1-activating region (SOAR) of Orai1. During this process, sensing Ca2+ by STIM1  induces the homo-oligomerization of STIM1 and its relocalization to the ER membrane near the plasma membrane where Oria1 is located, which also induces the homo-oligomerization of Orai1. The final hetero-oligomers that are formed by STIM1s and Orai1s are called ‘puncta’.In most cell types, the Ca122. There are several differences between SOCE in skeletal muscle cells and general SOCE in another cell types7. First, the SR or t-tubule in skeletal muscle corresponds to the ER or plasma membrane, respectively. Second, without Ca2+ depletion from the SR, puncta are formed during the development of skeletal muscle fibers and the process of terminal differentiation after birth 124. However, the prepuncta are not functional, and the additional conformational changes in Orai1 and/or STIM1 within the prepuncta are required to evoke SOCE123. An advantage of prepuncta formation in skeletal muscle is faster kinetics 126. In skeletal muscle fibers, SOCE is necessary to restore the SR with Ca2+ to the resting level and to sustain Ca2+ in the cytosol for skeletal muscle contraction during tetanic stimulation or fatigue128. In addition, it has been found that skeletal SOCE requires both structural and functional interactions with other proteins, such as MG29 , RyR1, and RyR3 in the triad junction129.The presence of SOCE in skeletal muscle was first identified in 2001131. Overexpression of CASQ1 in C2C12 myotubes inhibits SOCE102, while SOCE is increased in FDB skeletal muscle fibers from CASQ1 single- or CASQ1/CASQ2 double-knockout mice132. Therefore, CASQ1 not only modulates intracellular Ca2+ release but also provides a “reverse-directional signal” to regulate SOCE in skeletal muscle 133, suggesting that the functional interplay among CASQ1, RyR1 and SOCE could also contribute to pathological Ca2+ overload as well as to physiological Ca2+ signals. Indeed, SOCE is involved in muscle pathophysiology (see the next section on CASQ1-mediated diseases in this review)134.The fourth role of CASQ1 in skeletal muscle is as a regulator of SOCE via physical interaction between STIM1 and the acidic asp-rich/CAS region of CASQ1  interacts with two EC coupling-mediating proteins: the α1S subunit of DHPR via its cytosolic N-terminus and CASQ1 via its luminal C-terminus2+ entry through DHPR during skeletal EC coupling  is crucial to maintain muscle force development in the mouse slow-twitch soleus muscle, and ECCE is increased in FDB skeletal muscle fibers from JP-45/CASQ1 double-knockout, JP-45/CASQ2 double-knockout or JP-45/CASQ1/CASQ2 triple-knockout mice141, suggesting another type of ‘reverse-directional signal’ by CASQ1 via complex formation with JP-45. However, it is also controversial whether JP-45 affects ECCE through DHPR. Overexpression of JP-45 in C2C12 myotubes does not induce much change in the voltage-activated inward Ca2+ current 142. Therefore, more dedicated examinations are required to clarify the effects of JP-45 on DHPR or ECCE through DHPR in skeletal muscle.Ca2+ levels such as during skeletal muscle contraction or relaxation143. In cardiac muscle, IRE1a signaling plays protective roles under cardiac fibrosis or atherosclerosis145. These reports suggest the possibility that CASQ1 could participate in the stress-coping responses in skeletal muscle as well as cardiac muscle.CASQ1 prevents the dimerization of inositol-requiring enzyme 1α  by interacting with the luminal domain of IRE1α at JSR, which attenuates IRE1α signaling at physiological Ca2+-buffering proteins in the SR have been reported, such as histidine-rich Ca2+-binding protein  and sarcalumenin 147. The amounts of HRC and SLM in SR are much lower than the number of CASQ1109. Similar to CASQ1, HRC, and SLM are acidic and act as substrates of CKII. Unlike CASQ1, which is mainly located in the terminal cisternae of the SR, HRC, and SLM are more widely distributed and buffer Ca2+ throughout the SR148. The Ca2+-dependent phosphorylation of SLM and HRC by CKII inhibits the binding affinity of RyR1 to Ca2+149. This report suggests that HRC and SLM could regulate RyR1 in a Ca2+- or phosphorylation-dependent manner. In addition to CKII, HRC can be phosphorylated by Ca2+/calmodulin-dependent protein kinase II146. A 53-kDa Ca2+-binding protein is a splice variant of SLM150.Other Ca152. Calreticulin has no EF-hand consensus sequence; however, the highly acidic C-terminus of calreticulin accounts for its low-affinity and high-capacity Ca2+ binding properties.Calreticulin, also known as calregulin, is the ER homolog of CASQ1, and it also exists in skeletal muscle48, in the urinary bladder of guinea pig153, in Purkinje cells of chicken cerebellum154 and in the liver of rat155, but their functions have not been studied. A CASQ-like protein is found in body wall muscle cells of Caenorhabditis elegans (50% similarity and 30% identity to rabbit CASQ1), but it is not essential for body wall muscle formation and contraction156. CASQ-like proteins that are recognizable by anti-CASQ1 antibodies are found in the eggs of sea urchins (Strongylocentrotus droebachiensis and Arbacia punctulata)157.CASQ-like proteins have been found in other organisms or tissues, including in smooth muscles of the rat aorta and stomachStreptanthus tortuosus or spinach leaves158. The CASQ-like proteins in plants can bind to Ca2+ and are recognized by the anti-CASQ2 antibody. The other plant CASQ-like protein is found in Ca2+-accumulating cells from water lettuce (Pistia stratiotes)159. Various CASQ-like proteins in various types of cells and tissues from diverse species strongly suggest that the functions of CASQ1 could be more diverse than previously known.Interestingly, CASQ-like proteins have also been found in plants, such as cultured cells of 162. Patients with heterozygous missense mutations in the CASQ1 gene  show TAM164. I385T shows a slight increase in Ca2+-dependent tubular aggregation, but D44N, N56Y, and G103D show a decrease in Ca2+-dependent tubular aggregation. Interestingly, all the mutants reduced the amount of Ca2+ in the SR and D44N and I385T additionally showed a defect in the ability to inhibit SOCE163, again confirming that CASQ1 participates in the modulation of SOCE-mediated Ca2+ homeostasis via a reverse-directional signal.Tubular aggregate myopathy (TAM) was originally associated with mutations in STIM1 or Orai1, along with the dysregulation of SOCE CASQ1 gene) was the first identified CASQ1 mutant in patients showing mild myopathy characterized by muscle weakness, fatigue, and the presence of large vacuoles167. D244G induces misfolding and abnormal aggregation of CASQ1  due to the loss of the electric charge in D244 that is involved in the Ca2+-binding and Ca2+-dependent polymerization of CASQ1. Abnormal CASQ1 aggregates cause sarcoplasmic vacuolar aggregations (which induce protein aggregate myopathy (PAM)) and induce reduced Ca2+ release from the SR.D244G 172: C35-R614 in the N-terminus, D2129-R2458 in the central region, and I3916-G4942 in the C-terminus. MH is also attributed to mutations in the CACNA1S gene encoding the α1S subunit of DHPR173. In addition to the mutations in RyR1 and DHPR, sustained SOCE may affect the intracellular Ca2+ balance in the skeletal muscles of MH patients174. Until now, the only clinical treatment of MH has been the administration of dantrolene (a muscle relaxant)178. However, the molecular mechanism of the dantrolene effect has not been clearly demonstrated. What is the direct target of dantrolene? What proteins or pathways are involved in the dantrolene effect?Under the administration of volatile anesthetics such as isoflurane, sevoflurane, halothane, desflurane, and enflurane or muscle relaxants such as succinylcholine, patients with MH  can undergo sudden death by acute life-threatening skeletal muscle contracture with hypermetabolism that is characterized by increased OCASQ1 gene) show a mild association with MH167. M87T induces a mild reduction in the Ca2+-binding capacity of CASQ1 due to the shift of the M87-bearing α-helix and the weakening of the back-to-back interaction that is essential for the dimerization of CASQ1. CASQ1 knockout mice clearly show MH-like gain-of-function phenotypes that are very similar to those of RyR1 knock-in mice expressing human MH mutations 183, such as impaired movement, difficulty breathing, whole-body contractions, an arched back and finally rapid progression to sudden death. Loss of the inhibitory effect of CASQ1 on RyR1 activity and Ca2+ depletion from the SR due to the absence of CASQ1 explain the MH-like phenotypes in CASQ1 knockout mice119. A mutation in the CASQ1 gene  has been reported in MH patients, although its pathological mechanism has not yet been studied184. The locations of mutations that are related to patients with MH, TAM, or PAM are indicated in the 3D structure of human CASQ1 , resting free [Ca2+]cytosol and [Ca2+]SR is not significantly different between fast-twitch FDB skeletal muscle fibers from CASQ1 knockout and wild-type mice119. However, at >30 °C, [Ca2+]cytosol is significantly increased in FDB skeletal muscle fibers from CASQ1 knockout mice135. In addition, slow-twitch soleus muscle fibers from CASQ1 knockout mice are not significantly affected by temperature challenge135. These results from CASQ1 knockout mice suggest that the development of increased basal tension in fast-twitch muscle and the impaired control of [Ca2+]cytosol could be the starting point toward the development of contractures during MH or EHS crises  and/or strenuous exercise) exhibit remarkably similar symptoms to those in patients with MH188. First, structural remodeling of the triad junction, such as shrinking and abnormal multilayered triad junctions, occurs. Second, CASQ1 knockout induces the loss of inhibitory action of CASQ1 on RyR1 activity. Third, the absence of CASQ1 induces more rapid Ca2+ depletion from the SR due to the absence of Ca2+-buffering ability by CASQ1. Fourth, when exposed to halothane or heat, the knockout fibers show insufficient Ca2+ from the SR due to faster Ca2+ depletion from the SR with impairment of Ca2+ reuptake through SERCA1a into the SR. Fifth, more rapid Ca2+ depletion from the SR lowers the threshold for store overload-induced Ca2+ release (SOICR188). Sixth, the rapid Ca2+ depletion from the SR also increases skeletal SOCE to refill the SR. Seventh, the impaired SERCA1a activity probably increases ATP consumption, thus requiring a greater number of mitochondria . Eighth, all these abnormal events induce more increases in cytosolic [Ca2+]. Correlations between the abnormal events mentioned above and the causative roles of CASQ1 in MH in humans should be further investigated, although it has been revealed that each abnormal event is related to the characteristic muscle rigidity and rhabdomyolysism showed in patients with MH or EHS and death.Here is “a possible scenario” for the MH- or EHS-like phenomena in CASQ1 single- or CASQ1/CASQ2 double-knockout mice135. This sex-dependent mortality rate has also been found in human patients with MH or EHS190. The mechanisms responsible for the higher mortality rate in males have not been studied. A possible explanation for the sex-dependent mortality rate could be hormonal differences between males and females (related to different muscle masses and capacities).An unexpected property has been observed in CASQ1 knockout mice: a sex dependency. When CASQ1 knockout mice are exposed to halothane  or to high environmental temperature , male CASQ1 knockout mice exhibit a high mortality rate , but female CASQ1 knockout mice show only marginal effects191), dystrophic skeletal muscle fibers from a murine animal model of DMD  show reduced expression of CASQ-like proteins of 150–220 kDa 192. The decreased expression of CASQ-like proteins might be related to the altered Ca2+ level in the SR and cytosol and, subsequently, muscle necrosis or apoptosis in DMD patients. In addition, skeletal muscle fibers from another mdx mouse show excessive SOCE due to the increased expression of Orai1193, as shown in FDB muscle fibers from CASQ1 single- or CASQ1/CASQ2 double-knockout mice132. These findings suggest a strong possibility that, in addition to the main pathological role of dystrophin in DMD , abnormal Ca2+-buffering by CASQ1 and/or CASQ-like proteins in the SR, in combination with excessive SOCE, could be related to the pathological mechanism of DMD.Although the reduction or deficiency of dystrophin is the primary pathological cause of Duchenne muscular dystrophy  show increased mRNA levels of both the CASQ1 and CASQ2 genes in thyroid tissue197. Unlike normal subjects, patients with Graves’ hyperthyroidism without ophthalmopathy and Hashimoto’s thyroiditis with or without ophthalmopathy show significant levels of autoantibodies against CASQ1 in the serum199. The acute induction of hyperthyroidism in the rabbit soleus muscle increases the mRNA level of CASQ1200.Several other pathological conditions are related to CASQ1, although clear correlations between the pathological phenotypes of patients and CASQ1 have not been defined. CASQ1 is related to diabetes. A diabetic rat model has shown increased expression of CASQ1 with significantly increased Ca201.Although not discussed in this review, CASQ2 is closely related to human diseases, especially catecholaminergic polymorphic ventricular tachycardia . Regarding details on the relationship among CASQ2, RyR2, and CPVT in cardiac muscle, please refer to a review article2+-buffering protein in the SR of skeletal muscle, it is plausible that the knockout of CASQ1 in skeletal muscle can induce enormous reductions in Ca2+ release from the SR in response to various stimuli and in the amount of Ca2+ available for release from the SR; however, this is not observed (still significant but only relative reductions). In addition, only MH- or EHS-like symptoms, but not other skeletal muscle disease-like symptoms, have been found in CASQ1 single- or CASQ1/CASQ2 double-knockout mice. Muscle fibers from knockout mice still sustain a sufficient degree of Ca2+ release and muscle contraction. These curiosities raise the possibility that either CASQ1 has functions that are not currently understood or other proteins could compensate for the absence of CASQ1 alone or of both CASQ1 and CASQ2.For the previous three decades, extensive and dedicated studies on CASQ1 in skeletal muscle have been conducted, and the critical roles of CASQ1 have been revealed, which has been a monumental achievement in our understanding of the physiological and path-physiological properties of skeletal muscle. However, considering that CASQ1 is the major CaIn addition, no marked difference is observed between CASQ1 single- and CASQ1/CASQ2 double-knockout mice, which raises another important question: what is the physiological relevance of CASQ2 “in skeletal muscle”? It is possible that proteins and/or regulatory mechanisms exist, and further investigations of such proteins and/or mechanisms will help to answer these puzzling questions and improve our understanding of the physiological and pathophysiological properties of skeletal muscle.In our aging society, the number of patients with skeletal muscle diseases is continuing to increase, and improved understanding of the pathology of skeletal muscle is in great demand. A more detailed understanding of the pathological roles of CASQ1 in skeletal muscle, for example, the roles of CASQ1 in skeletal muscle diseases that are combined with other diseases, could be an excellent way to meet the demand.


STIM1 occupies space in the junctional SR membrane of the triads and the longitudinal SR at the Z-line. How STIM1 is organized and is retained in these specific subdomains of the SR is unclear. Here, we identified desmin, the major type III intermediate filament protein in muscle, as a binding partner for STIM1 based on a yeast 2-hybrid screen. Validation of the desmin-STIM1 interaction by immunoprecipitation and immunolocalization confirmed that the CC1-SOAR domains of STIM1 interact with desmin to enhance STIM1 oligomerization yet limit SOCE. Based on our studies of desmin-KO mice, we developed a model wherein desmin connected STIM1 at the Z-line in order to regulate the efficiency of Ca2+ refilling of the SR. Taken together, these studies showed that desmin-STIM1 assembles a cytoskeletal-SR connection that is important for Ca2+ signaling in skeletal muscle.Stromal interaction molecule 1 (STIM1), the sarcoplasmic reticulum (SR) transmembrane protein, activates store-operated Ca  STIM1 is best recognized as an activator of Orai channels, and together STIM1 and Orai1 channels form the basic elements for store-operated Ca2+ entry (SOCE)  is a multifunctional signaling protein in the sarcoplasmic reticulum/ER (SR/ER) membrane that can interact with many channels, transporters, and signaling molecules . Understy (SOCE) –5. Here,d SERCAs –10. How 2+ events appear to be limited to muscle development because the selective deletion of Orai1 from adult skeletal muscle results in no discernible phenotype  or selectively in skeletal muscle (mSTIM1–/–), display defects in skeletal muscle growth and reduced survival . We showed that desmin interacts with STIM1 at the CC1-SOAR domain where desmin can promote STIM1 oligomerization, restrict STIM1 migration, and delay SOCE activation. We used muscle fibers from mice lacking desmin to show that the presence of STIM1 near the Z-line was diminished in isolated flexor digitorum brevis (FDB) fibers, which reduced the efficiency of Ca2+ release channel in skeletal muscle, in the terminal cisternae  colocalized partially with ryanodine receptor 1 (RYR1) (red), the principal Caisternae . In addid Z-line . Togethehttps://doi.org/10.1172/jci.insight.143472DS1). The STIM1-LacZ fusion protein was able to insert into the SR membrane via an intact N-terminal domain and transmembrane segment, and an antibody to the N-terminus of STIM1 (green) was identical, as indicated by the line profiles  and Ca2+ refilling of the SR .To analyze the subcellular distribution of STIM1 in greater detail, we performed transmission electron microscopy (TEM) on skeletal muscles from heterozygous mice expressing a STIM1–β-galactosidase fusion protein (STIM1-LacZ) . The STIprofiles , C–H. Int muscle . Consistt muscle . Thus, S2+ stores . Partial clones encoding for desmin were detected as the interacting partner for STIM1. Desmin is a type III intermediate filament protein and the most abundant intermediate filament expressed in muscle. The terminal portion of the 2B region and the tail domain of mouse desmin (394–493 aa) represent the minimal region of desmin that interacted with STIM1 in the yeast 2-hybrid assay. Desmin was expressed early in the differentiation of C2C12 myoblasts and remained stably expressed throughout this period  19). ST. ST19). + stores  20). Bo. Bo2+ st+ stores , 22. To E. coli and purified it by glutathione column. Skeletal muscle lysates prepared from WT mice were applied to the column and the interaction was detected in the elute by immunoblotting for desmin. GST-STIM1 successfully pulled down endogenous desmin, but desmin did not bind to the control GST column  pulldown, co-IP, and immunostaining. First, we constructed a GST-STIM1 (251–685 aa) fusion protein in T column . We veriT column , lane 4.T column . To estaT column . CollectE. coli. The STIM1 peptides contained a His tag, which was used to purify the CC1-SOAR domain. When expressed by itself, the STIM1-CC1-SOAR peptides appeared as a 35 kD protein in the elute. Desmin was present in the elute of the STIM1-CT pulldown as an 11 kD band, as seen in To determine the peptide domains within STIM1 that mediate the desmin interaction, we tested whether specific STIM1 fragments containing a V5 tag were able to copurify with overexpressed desmin from HEK293 cells . To valiWe next tested the hypothesis that desmin associates with the carboxy-terminus of STIM1-SOAR  in order to influence STIM1 oligomerization, a mechanism important for STIM1’s actions on Orai1. We first examined whether STIM1-CT alone was sufficient for the interaction with desmin in vitro using a cell-free assay we described previously . Purifie2+ stores, STIM1 migrates continuously along microtubules viewed as discrete comets of ER-localized STIM1-GFP  experiments with STIM1-GFP. Desmin and STIM1-GFP colocalized in specific regions of the cell periphery of HEK293 cells . From th2+ signaling in vivo, we turned to DES-KO mice . We next measured cytosolic Ca2+ transients from Fura-4F-loaded FDB fibers in the absence of external Ca2+ and treated with cyclopiazoic acid (CPA) to block the SERCA1 pumping and caffeine to release RYR1 stores. Here, the AUC for the Ca2+ release was significantly reduced in the DES-KO fibers  indicator, D1ER, to measure free Ca2+ in the SR of these muscle fibers . Our results showed a small effect of tetracaine (1 mM) on the caffeine-induced store release for WT and DES-KO fibers, but this difference did not reach statistical significance. Moreover, SR Ca2+ stores from DES-KO were significantly reduced compared with WT fibers.  with a fast Ca2+ imaging system  was significantly prolonged in the DES-KO fibers that might represent reduced refilling of the SR  and Mg2+ (0.01 mM) to rapidly release Ca2+ stores as indicated by a reduction in fluorescence intensity of the Fluo-5N signal  or Ca2+ pump (SERCA1) levels in the DES-KO muscle  coupling, the well-established signaling complex that coordinates contraction. In addition, it has become increasingly clear that SOCE also contributes to muscle fiber Ca2+ dynamics and requires the assembly of a novel macromolecular complex , an endogenous inhibitor of SERCA2a, and likely contributes to spontaneous Ca2+ sparks  compared with WT mice. We attributed this effect to impaired activation of SOCE and reduced SR Ca2+ content  mice homozygous for the desmingenotype . STIM1+/r et al. .To isolate individual fibers, the FDB muscle was dissected from the foot carefully and immediately digested overnight at 37°C in digestion medium . After digestion, muscles were triturated in fiber medium (DMEM with 10% FBS and 1× Pen-Strep) until individual fibers separated. Fibers were plated onto glass-bottom plates coated with 20 μg/mL laminin). Fibers were allowed a period of 2 hours to recover before experiments were performed.Fibers were fixed in 4% PFA for 5 minutes, washed in PBS, and blocked in 10% heat-inactivated goat serum (HINGS). Fibers were incubated with primary antibody overnight at room temperature in fiber antibody solution . Primary antibodies are outlined in the See For ultrastructural localization of STIM1-LacZ by TEM, FDB muscles were fixed in situ for 5 minutes in 2% PFA 0.2% glutaraldehyde in PBS. Fibers were then dissected and stained for LacZ as described in Stiber et al. . Tissue HEK293 cells were cultured using standard protocols. C2C12 cells were maintained in growth media . To induce differentiation, the cells were switched from growth media to HEPES, insulin, and transferrin (HIT) media . Myotubes were used for experiments after 5 days in HIT media.g at 4°C, the soluble fraction was incubated with the appropriate antibodies at the specified dilution overnight. For GST pulldowns, STIM1 fragment (251–493 aa) was cloned in frame downstream of the GST epitope. GST or desmin-GST plasmid was transformed into E. coli and the GST-desmin protein was affinity-purified on the glutathione column. Muscle lysates from WT and STIM1-KO mice were prepared as above and passed through the column. After extensive washes, specific elutions were collected and subjected to SDS-PAGE. Immunoblotting with GST or STIM1 antibodies were used for detection of binding.Uncut gels can be viewed in the online supplemental material. Protein lysates were prepared and solubilized from gastrocnemius skeletal muscle harvested from mice using a standard RIPA buffer  supplemented with protease and phosphatase inhibitor cocktail. Lysates were homogenized and incubated on ice for 30 minutes. After centrifugation at 15,000g for 30 minutes. Supernatant was loaded on a pre-equilibrated cobalt bead column, washed with the above buffer twice, buffered with 25 mM imidazole once, and then eluted with elution buffer (binding buffer with 300 mM imidazole). The supernatant and elution were loaded to 16% gel for detection.Desmin-CT (371–470 aa) and STIM1-CT were cloned into pETDUET-1 (Novagen). The verified plasmid was transformed into BL21 for overexpression overnight at 23°C with 1 mM IPTG induction. Cells were harvested and resuspended in binding buffer . Cells were sonicated and centrifuged at 4°C, 10,000Plasmids expressing STIM1 fragments with a V5 tag were coexpressed with desmin in HEK293 cells. V5 or desmin antibodies were incubated with cell lysate and enriched to protein A/G beads and eluted by loading buffer after sufficient washing with the same amount of IgG as the negative control. Western blotting was performed by resolving proteins at different percentage SDS-PAGE gels according to molecular weight, transferred to PVDF membrane, and then blotted with specific antibodies.C2C12 cells were cultured and differentiated as described above. Cell lysates were prepared in RIPA buffer in PBS (pH 7.4). Next, 100 μg total protein was used for cross-linking with DTBP (5 mM) at room temperature for 30 minutes. For a negative control, proteins were treated with DTBP (5 mM) and DTT (10 mM) at room temperature. Lysates were separated by SDS-PAGE and immunoblotted for desmin or STIM1 as described above.cDNA sequence encoding the C-terminus of STIM1 (238–535 aa) was subcloned into the DW464 vector, which was then used to make baculovirus that expressed biotin-labeled STIM1c. Biotin-tagged STIM1c was purified using the BioBac Sf9 baculovirus purification system. Sf9 infected with STIM1c baculovirus was harvested 72 hours later by centrifugation at 448 RCF for 5 minutes (4°C). The pellet was washed in PBS and lysed in lysis buffer . The lysate was then loaded on to the NeutrAvidin affinity column (Pierce). The column was first washed 3 times in buffer A  and then 3 times in buffer B . Purified protein was eluted from the column by elution buffer .3, pH 8.5 overnight at 4°C. The wells were then blocked by 2% milk in 100 mM NaHCO3, pH 8.5 at room temperature for 1 hour. The wells were washed by PBST and 4 pmol of purified biotin-STIM1c protein was then added into each well and incubated overnight at 4°C. Negative control wells were coated with 2% milk in PBS. Skeletal muscle lysate or HEK293 cell lysate that contained overexpressed His-tagged Orai1 was incubated with the STIM1-CT–coated wells for 2 hours at room temperature and washed 5 times by PBST. Anti-desmin, anti-His tag, anti-TRPC1 , and anti-STIM1 N-terminus antibodies were then incubated with the wells at the dilution of 1:1000 at room temperature for 2 hours. The wells were washed 5 times with PBST and incubated with 1:3000 secondary antibodies conjugated with HRP for 1 hour at room temperature. The wells were then washed with PBST 5 times and incubated with ABTS with 0.05%H2O2 at room temperature for 10 minutes, and the plate was read at 405 nm with a plate reader.Costar 96-well plates were coated with NeutrAvidin in 100 mM NaHCO2+ quench experiments, cytosolic Ca2+ indicator Fura-2 (2 μM) was loaded into muscle fibers plated on 35 mm MatTek dish at 37°C for 45 minutes in zero Ca2+ Tyrode solution including (in mM) 145 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1 MgCl2, 5 HEPES, and 10 glucose, pH 7.4; SR Ca2+ was depleted with 30 μM CPA while loading dye. Next, 50 μM BTS was used to inhibit muscle contraction and reduce the motion artifact during Ca2+ imaging. Fura2-loaded FDB muscle fiber was then mounted to a motorized Nikon eclipse TE 2000-E microscope stage. The microscope was equipped with a Nikon 40× oil-based objective. The FDB fibers were continuously perfused with oxygenated experimental solutions. Epi-fluorescence of Fura-2 was excited by a pE-340fura illumination system (CoolLED). Ca2+ images were recorded with a PCO Edge 5.5 digital camera. The imaging data were acquired with MetaFluor software (Molecular Devices). After a stable baseline was recorded in the SR depletion solution, a Mn2+ quenching solution that contained 30 μM CPA, 2 mM Ba2+, and 0.5 mM Mn2+ was applied to activate SOCE and to quench fura-2 fluorescence. SOCE was measured as the initial slope of decrease in fura-2 fluorescence. For SR store Ca2+ content measurement, a low-affinity Ca2+ indicator, fura-4F, was used to avoid possible saturation. Next, 2 μM fura-4F was loaded in normal Tyrode solution for 45 minutes at 37°C. After 5 minutes of baseline recordings, SR store Ca2+ was depleted by Tyrode solution of 0 Ca2+, 10 mM caffeine, 10 μM verapamil, and 30 μM CPA. SR store Ca2+ content was calculated by integrating the area under the Ca2+ release curve.For Mn2+ images of a single fiber were captured with a PCO Edge 5.5 digital camera at a rate up to 500 fps and acquired with micromanager software. Ca2+ imaging data were analyzed with ImageJ (NIH). The decay time of fluorescence immediately after a 2-second field stimulation pulse was exponentially fitted with an order of 2, defined as f(t) = A1e–t/τ1 + A2e–t/τ2 + C.FDB fibers were loaded with 5 μM Fluo-3 for 30 minutes in a 37°C incubator. Next, 5 mM Fluo-3 stock was reconstituted at 20% Pluronic in DMSO. Electrical field stimulation of a single FDB fiber was generated by an A310 Accupulser (World Precision Instruments) and an A385 Stimulus Isolator (World Precision Instruments) connected to the insert for a 35 mm MatTek glass-bottom dish. A 2-second duration of bursts of stimulation consisting of 1 ms current pulses (100 mA) was applied at 50 Hz. CaFRAP was performed with a Prairie confocal system equipped with a photoactivation/FRAP module and an Olympus BX51Wi microscope with a 60× water immersion objective. HEK293 cells transiently transfected with GF-tagged STIM1 were plated in a MatTek 35 mm glass-bottom dish. GFP fluorescence was excited with a 488 nm laser. The region of interest (ROI or mask) was defined using Prairie view software and then photobleached with a 405 nm laser for 50 ms at maximal laser power. Fluorescence recovery of ROI after photobleaching was recorded continuously for 50 seconds. The intensities of fluorescence in the photobleached area were analyzed and normalized to that before photobleaching.2+ indicator was electroporated into FDB fibers of 2-month-old FVB (CTL) and DES-KO mice. Then, 10 to 14 days after electroporation, single FDB muscle fibers were isolated using the enzyme digestion method. Fibers were plated to a glass-bottom 35 mm MatTek dish and mounted to a Nikon TE2000-E microscope for Ca2+ imaging. The D1ER was excited with a 436 nm LED light (CoolLED). Cyano and yellow fluorescent proteins  fluorescence were filtered with narrow-band FRET filters installed inside a motorized cubic filter wheel and collected with a 250 ms delay. Ca2+ imaging data were recorded and analyzed with MetaFluor software (Molecular Devices). SR Ca2+ was released using a 0 mM Ca2+ Tyrode solution containing 30 mM caffeine. The contraction of muscle fiber was inhibited with myosin inhibitor BTS (50 μM).The plasmid encoding the D1ER SR Ca2+ with Fluo-5N fluorescence, as previously described  50 EGTA, 36 NaCl, 10 HEPES; 126 K+, 8 ATP, 10 creatine phosphate, and 1 Mg2+, pH 7.2. Cytosolic free Ca2+ concentration was calculated with a web-based program (http://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/webmax.htm). Next, 0–42 mM CaCl2 was added to standard internal solution as previously described . Emission of fluorescence was collected and recorded with a PCO Edge camera controlled by MetaFluor software (Molecular Devices). Ca2+ imaging data were further analyzed after subtraction of background fluorescence and normalized with F/F0.To assay SR Caescribed , isolateescribed . Ca2+ wan (mouse and muscle tissue) for each group was between 8 and 20 to achieve a statistical power of 80% (beta = 20%). Fibers (WT vs. KO) were allocated randomly after isolation for Ca imaging and histology; otherwise, no randomization was performed. The principal operator was blinded to the genotypes for calcium imaging and histology. Comparisons were made among gender- and age-matched controls for several independent litters. No data were excluded from analysis. All data points represent biological replicates. Population means were tested for significance using a Student’s 2-tailed t test. A P value less than 0.05 was considered significant.Averaged results are presented as mean ± SEM. Based on a power calculation using 95% CI and assuming a 20% difference in the magnitude of the effect between groups, a sample size of Yeast-2 hybrid was performed by PBR. TL, JPK, and C Wang conducted the STIM1 biochemistry and molecular studies. Mice were bred and maintained by VGB, RW, and HZ. Immunofluorescence was performed by VGB and JPK. TEM studies were performed by VGB. Calcium imaging was performed by HZ and C Ward. PBR and C Ward interpreted the data and wrote the manuscript. DMM and RJB contributed to interpretation and writing of manuscript. All authors contributed to editing of the manuscript and figures.


Therefore, intestinal anion secretion from native mouse duodenal epithelia was examined with Ussing chambers to monitor PGE2-, 5-HT-, and CCh-induced short-circuit currents (Isc). PGE2 (10 μM) and 5-HT (10 μM) induced mouse duodenal Isc, markedly attenuated by serosal Ca2+-free solution and selective blockers of store-operated Ca2+ channels on the serosal side of the duodenum. Furthermore, PGE2- and 5-HT-induced duodenal Isc was also inhibited by ER Ca2+ chelator TPEN. However, dantrolene, a selective blocker of ryanodine receptors, inhibited PGE2-induced duodenal Isc, while LiCl, an inhibitor of IP3 production, inhibited 5-HT-induced Isc. Moreover, duodenal Isc response to the serosal applications of both PGE2 and 5-HT was significantly attenuated in transient receptor potential vanilloid 4 (TRPV4) knockout mice. Finally, mucosal application of carbachol (100 μM) also induced duodenal Isc via selective activation of muscarinic receptors, which was significantly inhibited in serosal Ca2+-free solution but neither in mucosal Ca2+-free solution nor by nifedipine. Therefore, the serosal TRPV4-constituted SOCE mechanism is likely universal for the most common and important secretagogues-induced and Ca2+-dependent intestinal anion secretion. These findings will enhance our knowledge about gastrointestinal (G.I.) epithelial physiology and the associated G.I. diseases, such as diarrhea and constipation.As little is known about the role of calcium (Ca Since water follows ion movement across osmotic gradients, which is primarily generated by chloride ion  classically. The intracellular store in the endoplasmic reticulum (E.R.) from which Ca2+ is released in two main ways, which is via the ryanodine receptor (RyR) or the inositol trisphosphate receptor (IP3R) (2+ release-activated Ca2+ channels (CRAC) were first described in mast cells and Jurkat lymphocytes  . These Cphocytes . Howeverlucidate . In addi3R/ER Ca2+ release, but caffeine triggered RyR/ER Ca2+ release, both of which stimulated serosal store-operated Ca2+ entry (SOCE) mechanism and eventually induced Ca2+-dependent duodenal anion secretion  if serosal SOCE is a universal mechanism for Ca2+-dependent duodenal anion secretion; 3) if so, what molecular components of the SOCE are involved in this process; and 4) if CCh evokes a Ca2+-dependent anion secretion when applied from the mucosal side of the duodenum, although it is well known to stimulate it from the serosal side. Therefore, we aimed to investigate these important issues using native duodenal epithelial tissues in mice as a follow-up study.We previously demonstrated that carbachol (CCh), a stable chemical analog of neurotransmitter ACh, triggered IPecretion  However,2. Animals were assigned randomly to different experimental groups of all studies. Data collection and evaluation of all experiments performed blindly, and the experimenters were unaware of group treatments.All experiments were adopted with adult male Harlan C-57BL/6 mice  and transient receptor potential vanilloid 4 (TPPV4) deficient (TRPV4 KO) mice which generated from C-57BL/6 mice . Animal care and experiments conformed with the guidance of the Animal Ethical Committee of the University and were approved by the University Committee on Investigations Involving Animal Subjects. According to the ARRIVE guidelines , the mic2+]cyt measurement.IEC-6, a small intestinal epithelial cell line of rat origin , was obt2, 1.2 mM MgCl2, and 10 mM D-mannitol at pH 7.4 when gassed with Oxygen (100% O2) at 37°C. The serosal solution contained the following: 115 mM NaCl, 25 mM NaHCO3, 2.2 mM K2HPO4, 1.2 mM CaCl2, 1.2 mM MgCl2, 0.8 mM KH2PO4, 10 mM D-glucose, and this solution was gassed with carbogen  at 37°C and had a pH 7.4. For the Ca2+-free experiments, Ca2+ was omitted, and EGTA (0.5 mM) was added in both mucosal and serosal solutions to prevent potential Ca2+ contamination. The physiological salt solution (PSS) used in digital Ca2+ measurement contained the following: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4). For the Ca2+-free PSS solution, Ca2+ was omitted, but 0.5-mM EGTA was added. The osmolalities for all solutions were 300 mOsmol kg−1 of H2O.Solutions to the mucosal side in the Ussing chamber experiments contained the following: 115 mM NaCl, 25 mM sodium-D-gluconate, 5.2 mM potassium-D-gluconate, 1.2 mM CaCl2, which is resulting from mucosal injury during experiments, to avoid affecting the basal Isc. Finally, the section is opened longitudinally, with the mesenteric attachment remnant, seromusculature stripped, and divided into four segments. The segment, which is likely to undergo less excision damage, will be situated in the chambers’ aperture .Following euthanasia, the mice’s abdomen was opened by a midline incision. Next, we dissected the proximal duodenum 4 cm from the pylorus carefully and immediately but not pulled to avoid damaging the epithelium. Afterward, the duodenum section was incubated in ice-cold iso-osmolar mannitol (300 mM) and indomethacin (1μM) solution 10 min before seromuscular stripping to inhibit possible endogenous PGEIsc), while μA was used for the original recordings, and μA cm−2 was used for summary data. After 10–15 min of measurements for basal parameters, various agonists or antagonists were added to one side or both sides for 10–20 min, followed by PGE2, 5-HT, and carbachol.Segments were fixed in a modified Ussing chamber bathed with a volume of 3 ml on each side of the mucosa preparation at 37°C and short-circuited by a computer-controlled voltage-clamp device  under continuous short-circuited conditions. An automatic voltage-clamp measured the transepithelial short-circuit currents , SKF-96365, TPEN  ethylenediamine), GSK-7975A, ouabain, HC067047, and GSK1016790A were purchased from MedChemExpress . Sigma  supplied carbamylcholine chloride (CCh), nifedipine, gadolinium chloride, and cyclopiazonic acid (CPA). Tocris Bioscience  supplied 2-Aminoethoxydiphenyl borate (2-APB), while APExBIO Technology LLC  provided dantrolene. Fura-2 was purchased from Invitrogen , meantime DMEM and FBS were obtained from Hyclone . Trypsin and penicillin/streptomycin were purchased from Gibco . The other chemicals were obtained from BBI Life Science .Prostaglandin En) of investigated tissues. Net peak of duodenal Isc refers to drug-stimulated maximal peak minus basal level. The statistical significance of differences in experimental groups’ means was determined by using Student’s unpaired, two-tailed t-test or one-way ANOVA followed by Dunnett’s post-test. Post hoc tests were run if F achieved p < 0.05 (GraphPad Prism 8.0), and there was no significant variance in inhomogeneity. A probability P-value <0.05 was considered statistically significant.Data and statistical analysis yield to the recommendations of Frontiers in Pharmacology. All results are given as means ± standard error of the mean number  in the serosal induced a transient high Isc peak with a sustained phase following (2 and vehicle (DMSO) mucosal application did not affect the duodenal Isc  in the serosal side significantly reduced PGE2-stimulated duodenal Isc  can mediate Ca2+ release from E.R., serosal addition of dantrolene (300 μM), a selective RyR antagonist, markedly inhibited PGE2-induced Isc  also leads to E.R. intracellular Ca2+release through E.R. membrane by IP3 receptors  ethylenediamine (TPEN) can rapidly and reversibly chelate Caith Ca2+ , which wced Isc , suggesteceptors , we usedced Isc . These f2-stimulated duodenal Isc. We all know that GSK1016790A, a highly selective agonist of TRPV4, can activate TRPV4 in diverse cells  serosal addition did not affect the PGE2-stimulated duodenal Isc of TRPV4 knockout mice. Above all, our findings are suggesting that TRPV4 is the molecular constituent of CRAC channels.Since that 2-APB, SKF-96365 and GSK-7975A also act on the transient receptor potential V(TRPV) family and that TRPV4 channels are expressed in the G.I. tract , we testse cells . Howeverls , we tested if it is involved in the process of PGE2-stimulated duodenal Isc. Because NKA is exclusively expressed at the serosal side in the intestinal epithelium , was added to the serosal side, duodenal n 10 min . However tissues . Therefo2+-dependent duodenal ion secretion, we examined if the above mechanisms similar to PGE2 are involved. First, we utilized three antagonists of SOCE. 2-APB (100 μM) application to the serosal side significantly suppressed 5-HT-evoked Isc, but the mucosal side application did not affect Isc  was added to the serosal side, 5-HT-stimulated duodenal Iscis markedly inhibited in duodenal tissue (3 (30 μM) application on the serosal side also significantly reduced 5-HT-stimulated duodenal Isc  serosal addition did not affect the 5-HT-stimulated duodenal Isc of TRPV4 knockout mice  to serosal side of the duodenum and found that TPEN significantly inhibited 5-HT-stimulated Isc (2+ store in this course. Then we used LiCl (30 mM) to inhibit IP3 production but dantrolene (300 μM) to inhibit RyR. Interestingly, we found LiCl but not dantrolene significantly inhibited 5-HT-induced duodenal Isc , an L-type calcium channel blocker, did not inhibit luminal CCh-induced Isc either  in the serosal side to test if it affects the luminal CCh induced Ca2+-dependent duodenal Isc. As shown in 3+ significantly reduced luminal CCh-stimulated duodenal Isc, which suggested luminal CCh evokes anion secretion through SOCE mechanisms.As Gd2-stimulated duodenal Isc between wild-type and TRPV4 KO mice. As shown in Isc induced by serosal addition of luminal CCh was significantly attenuated in TRPV4 KO mice. Meanwhile, GdCl3 (30 μM) serosal addition did not affect luminal CCh-stimulated duodenal Isc of TRPV4 knockout mice  in the serosal side significantly reduced CPA-stimulated duodenal Isc (Isc peak with a sustained phase following (3 (30 μM) and HC067047 (30 μM) in the serosal side significantly reduced TPEN-stimulated duodenal Isc , an ER-Canhibitor , can actnhibitor , TPEN alhin E.R. . The addollowing , and thenal Isc . The addollowing , while tnal Isc .2+]cyt rising were suppressed by pretreatment with RN1734 or extracellular Ca2+ omission in IEC-6 cells (2+]cyt in IEC-6 cells to test the TRPV4 constituent SOCE mechanism. Superfused with Ca2+-free solution (0 Ca), CPA (10 μM), and TPEN (50 μM), first induced a rapid increase in [Ca2+]cyt in IEC-6 cells as a result of ER Ca2+ release  caused an enhanced increase in [Ca2+]cyt owing to the SOCE mechanism  CARC may act as the SOCE mechanism in the process of Ca2+-dependent anion secretion. 3) TRPV4 channels may represent the molecular constituents of serosal SOCE/CARC channels to mediate Ca2+-dependent anion secretion. Therefore, our results indicate that Ca2+ signaling is essential for three most common and important secretagogues-induced small intestinal anion secretion, in which serosal TRPV4-constituted SOCE mechanism may play a critical role. Thus, our findings supple a novel insight into the molecular mechanisms of secretagogues-mediated epithelial anion secretion via Ca2+ signaling.Albeit it is well recognized that the critical role of calcium signaling in epithelial ion transports of the salivary gland, pancreatic ducts, and colonic epithelia, the detailed regulatory in the small intestinal epithelial and the underlying molecular mechanisms are not fully understood. Our research demonstrates that: 1) serosal PGE2+]cyt serve to regulate numerous cellular functions in various mammalian cells  LiCl, an inhibitor of IP3 production attenuated 5-HT-induced but not PGE2-induced Isc. These findings suggest that ER Ca2+ release may be induced via different pathways: PGE2 and 5-HT activate RyR and IP3R, respectively. Although ER Ca2+ release is mainly mediated by two well-known channels RyR and IP3R, they have different Ca2+ affinities  dose-dependently enhanced the inhibition. However, whether the high concentration of dantrolene has a non-specific effect needs further investigation in RyR KO mice or cell lines. Moreover, 2-APB is a non-specific IP3R inhibitor, so we chose to examine the involvement of IP3R by inhibition of IP3 with LiCl. Further experiments in K.O. mice or cell lines may be needed to verify IP3R involvement in Isc and SOCE further.Furthermore, we reveal a difference between them: 1) dantrolene, a selective RyR antagonist, inhibited PGEfinities . 5-HT co release . In contry cells . The conry cells  that ser2- and 5-HT-induced Ca2+-dependent duodenal anion secretion for the first time. In the meantime, we adopted a TRPV4 blocker to suppress SOCE activation in the IEC-6 cell line, which elucidates that TRPV4 participates in SOCE. However, GSK1016790A alone had no effect on the basal Isc in W.T., and K.O. mice, which could be due to the fact that directly opening TRPV4 channel itself may not be sufficient to cause calcium influx in situ because TRPV4 is only one of many components for SOCE activation, including STIM1, Orai, TRPC channels.Molecular components of SOCE in intestinal epithelial cells have not been well identified. They are considered as TRPC1 in IEC  or STIM1Although the role of TRPV4 channels in G.I. disease has been broadly examined , their p2+ signaling mediated anion secretion by TRPV4-constituted serosal SOCE mechanisms, which is universal for the three most common and important secretagogues. Although PGE2 and 5-HT stimulate this mechanism exclusively from serosal side of the duodenum, CCh stimulates it from both sides. We also reveal that 5-HT and CCh trigger ER Ca2+ release to initiate SOCE likely via IP3/IP3R, but PGE2 triggers it likely via RyR. A diagram is depicting, which we find in 2+ signaling will enhance our knowledge of G.I. epithelial physiology and G.I. disease associated with abnormal anion secretion, such as diarrhea and constipation.In conclusion, we underscore an essential role of Ca


Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications. Two funa crassa ] and Carujikuroi ] rhodopsmaculans ] rhodopsmaculans . While Lmaculans . While tmaculans .Microbial rhodopsins play an important role in optogenetics, allowing for the light-controlled manipulation of neuronal functions . Genes cCaenorhabditis elegans   . As a prfections .A. pullulans, three putative rhodopsin genes are found, ApOps1 (gb|KEQ89910.1), ApOps2 (gb|KEQ89333.1), and ApOps3 (gb|KEQ87154.1). ApOps1 and 2 are CarO-like rhodopsins, while ApOps3 is assigned to LR-like rhodopsins , CarO (gb| CAD97459), LR (gb| AAG01180), ApOps1 (gb|KEQ89910.1), ApOps2 (gb|KEQ89333.1), and ApOps3 (gb|KEQ87154.1) were aligned using PSI/TM-Coffee alignment  and random hexamer primers (Promega) according to the manufacturer instructions. Coding sequences of ApOps1 and ApOps2 were amplified from cDNA by PCR. ApOps3 was synthesized by ProteoGenix (France). ApOps1 and ApOps2 were cloned into pcDNA5/FRT/TO-CarO::eYFP ] were inserted via BglII and XhoI restriction sites into the pGEMHE vectors already containing the cleavable N-terminal signal peptide Lucy-Rho (LR), eYFP, the plasma membrane trafficking signal (T), and the ER export signal (E). Mutations were made by QuikChange site-directed mutagenesis. The generated plasmid sequences were confirmed by DNA sequencing. The linearized plasmids (by NheI digestion) were used for in vitro generation of cRNA with an AmpliCap-MaxT7 high-yield message maker kit (Epicentre Biotechnologies). Afterward, the DNA fragments coding for proton pumps or channels together with fused signal peptides and eYFP were transferred to plasmid pcDNA3.1 (-) via BamHI and HindIII restriction sites for mammalian cell expression.The PCR-amplified DNA fragments of ApOps1-3 and CsR  yield 1via a fluorescent protein and the beta-subunit of the H+/K+-ATPase with the second rhodopsin  in the C-terminal position  to histidine (H) happened during the cloning process. Since the R position is mostly conserved in the transmembrane helix 3 of different rhodopsins and it is known that it is of importance for the proton transfer, we decided to keep the wrong clone/mutant for a measurement. We found it to be a light-gated proton channel . We used site-directed mutagenesis to introduce the similar mutation R112H to ApOps2 and also the corresponding mutations in ApOps1 and ApOps3 , and pHi and pHe represented intracellular and extracellular pH, respectively  in comparison to ApOps2 2.0  and ApOps3 2.0 . The rhodopsin density was in a similar range as previously described for HEK293 cells expressing ChR2-eYFP  is striking, even for extremophils that can be exposed to very strong radiation. Nevertheless, the absence of saturation at a value of 20 mW mm−2 was previously observed in eArch3.0, a proton pump from the halophilic archaeon Halorubrum sodomense (Chlamydomonas rheinhardtii that concentrate the light and focus it onto the receptors, which would lead to much higher light intensities (The absence of saturation even at very high light intensities that are more than 10-fold above typical sun radiation (∼1 mW mmodomense . It is qensities . On the ensities . When in


This arrangement in turn ensures efficient triggering of Ca2+ release, and contraction. While new data indicate that t-tubules are capable of exhibiting compensatory remodeling, they are also widely reported to be structurally and functionally compromised during disease, resulting in disrupted Ca2+ homeostasis, impaired systolic and/or diastolic function, and arrhythmogenesis. This review summarizes these findings, while highlighting an emerging appreciation of the distinct roles of t-tubules in the pathophysiology of heart failure with reduced and preserved ejection fraction (HFrEF and HFpEF). In this context, we review current understanding of the processes underlying t-tubule growth, maintenance, and degradation, underscoring the involvement of a variety of regulatory proteins, including junctophilin-2 (JPH2), amphiphysin-2 (BIN1), caveolin-3 (Cav3), and newer candidate proteins. Upstream regulation of t-tubule structure/function by cardiac workload and specifically ventricular wall stress is also discussed, alongside perspectives for novel strategies which may therapeutically target these mechanisms.In cardiomyocytes, invaginations of the sarcolemmal membrane called t-tubules are critically important for triggering contraction by excitation-contraction (EC) coupling. These structures form functional junctions with the sarcoplasmic reticulum (SR), and thereby enable close contact between L-type Ca Forceful contraction of the heart requires coordinated contraction of cardiac muscle cells, called cardiomyocytes. Within these cells, electrical excitation during the action potential is linked to cell shortening by a process known as excitation-contraction (EC) coupling. In mammalian heart, this process is made possible by membrane invaginations called t-tubules which propagate the action potential into the cell interior. Critical in normal cardiac physiology and importantly disrupted during disease, t-tubules have been the focus of considerable research focus in the past decades. This review will summarize this progress and outline future perspectives, with particular attention given to potential roles of t-tubules as therapeutic targets.A schematic overview of cardiomyocyte t-tubule structure is provided in There are considerable species differences in t-tubule density and organization . Indeed,2+ channels ; a process called Ca2+-induced Ca2+ release (CICR) , or extruded from the cell by the Na+/Ca2+ exchanger (NCX), and to a lesser extent, the sarcolemmal Ca2+ ATPase  deep into the cell interior, and the resulting depolarization of the t-tubular membrane leads to opening of voltage-gated L-type Cae (CICR) . Binding+ ATPase .2+ release across the cell. This is particularly apparent in mouse and rat ventricular cardiomyocytes, due to the robust presence of these structures , or a “super-cluster,” with released Ca2+ jumping between nearby clusters to produce a Ca2+ spark , junctophilin-2 (JPH2), and caveolin-3 (Cav-3) , which aThe process of t-tubule development varies between species . In spec2+ transient from the surface of the cells to the interior  is a leading cause of death worldwide. It is therefore of great importance to understand the different mechanisms underlying this condition, to facilitate discovery of novel treatment targets. HF can be divided into two main entities; HF with reduced ejection fraction (HFrEF) and HF with preserved ejection fraction (HFpEF). HFrEF describes a state where the cardiac muscle is unable to contract adequately, leaving the heart unable to meet the body’s oxygen demand. This state is associated with ventricular dilation, thinning of ventricular walls and high wall stress. HFpEF, on the contrary, is predominantly linked to cardiac hypertrophy, wall thickening, and maintained wall stress . Althougper se. Further complicating the picture is the observation that t-tubule remodeling in HFrEF frequently includes an increased fraction of longitudinally oriented tubules  results, while delayed afterdepolarizations (DADs) are triggered by spontaneous Ca2+ release between beats  RyRs, and resulting increased Ca2+ influx has been predicted to contribute to SR Ca2+ overload  . Of thesAs In summary of the above discussion, it appears to be no coincidence that the vast majority of present HFrEF therapies relieve symptoms and delay disease progression by reducing the workload of the heart, and specifically the physical stress placed on the ventricular wall . However2+ release from early developmental states, impaired release Ca2+ release was noted along t-tubules in failing cells  critically regulates tubulogenesis . This me2 (A key aspect of Bin1’s function appears to be its ability to cluster phosphoinositides (PIs), in particular PI4, 5P2 . The dom2 . This pr2 . Althoug2  and MTM12 . Collect2 , 5.As noted in the first chapter, JPH2 is a membrane anchoring protein, important for connecting the sarcolemma and its t-tubules to the SR in the dyad . In thisAs with BIN1, JPH2 has been shown to play a key role in the development of t-tubules and dyads, both pre- and postnatally . This fu2+ release, and HFrEF , as What causes JPH2 changes during disease? A suggested mechanism for suppression of JPH2 expression is Calpain cleavage of JPH2 is believed to be one of the mechanisms behind its downregulation, as first reported in mouse models of reversible heart failure , and iscFinally, the function of JPH2 has been shown to be dependent on its phosphorylation status , which iTaken together, there is an abundance of evidence to support that JPH2 is a critical regulator of dyadic assembly and maintenance, and that this function extends far beyond a passive role in anchoring membrane positions. Thus, JPH2 is a promising target for future therapies.2+ current density and impairment of Ca2+ release, linked to lowered levels at Cav-3  . Previoueostasis . These feostasis . Furthereostasis .via necrosis (The muscle-specific membrane repair protein mitsugumin 53 (MG53) is an up and coming t-tubule regulator . This “wnecrosis . Regulatnecrosis . When annecrosis . MG53 conecrosis . He et aProtein kinase C (PKC) activation has been implicated in t-tubule remodeling in a recent study by via stretch-activated channels) or Z-disks (T-Cap), or are linked to the mechanosensitive process of actin polymerization . A perhaps somewhat distinct group of t-tubule regulatory proteins appears to be focused on maintenance of the membrane itself, and notably includes Mtm1, BIN1, and the general autophagic and endocytic processes. It is less clear that these processes are mechanosensitive in cardiomyocytes, and we might rather speculate that the changing metabolic environment of the diseased heart is an overarching signal that regulates membrane integrity.The above discussion has highlighted recent work implicating a plethora of proteins involved in the growth and maintenance of t-tubules and their remodeling during disease. How should we make sense of this increasingly complex array of proteins? Are there shared, overarching signaling pathways which coordinate changing protein expression patterns? We believe that changing physical stress experienced by the myocardium is at least one such signal. Indeed, when viewed from a mechanosensatory viewpoint we can see that many of the proposed t-tubule regulators can be functionally clustered together. For example, several of these proteins have proposed roles in mechanosensing at the cell membrane (PKC Despite promising results from rodent studies, it seems unlikely that targeting of only a single t-tubule regulator would be sufficient to therapeutically protect t-tubule structure in humans. Rather, we believe that new interventions should instead be aimed at the overarching signals and functional groups of proteins described above. For example, upregulating expression of JPH2 has been suggested as an approach to maintain dyadic structure during HFrEF . However2+ homeostasis, and thus systolic and diastolic function of the heart as a working organ. The discussion has highlighted a consensus view that t-tubule remodeling is a key mechanism contributing to disrupted Ca2+ handling and contractility in HFrEF, and a likely contributor to arrythmogenesis, but that distinct forms of remodeling occur during HFpEF. We have described a wealth of evidence indicating key roles of dyadic proteins JPH2, BIN1, and Cav-3, but also newer players and signaling pathways which hold promise. We believe that several current HFrEF therapies preserve t-tubule structure and cardiac function by normalizing expression of these proteins, and that this involves relief of the high workload that drives dyadic disruption. We anticipate that improved safeguarding of t-tubule integrity will serve as a basis for future HFrEF therapy.This review has summarized our growing appreciation for the role of t-tubules and dyads as critical regulators of cardiomyocyte CaAll authors contributed to the writing of the article and development of the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.