RAS or FLT3 are more typically seen in AML or otherwise are a harbinger of transformation. In this review, we focus on the insights into the biological and genetic distinctions and similarities between MDS and AML that are now used to refine clinical prognostication, guide disease management, and to inform development of novel therapeutic approaches.Myelodysplastic syndrome and acute myeloid leukemia are heterogeneous myeloid neoplasms which arise from the accumulation of mutations in a myeloid stem cell or progenitor that confer survival or growth advantages. These disease processes are formally differentiated by clinical, laboratory, and morphological presentations, especially with regard to the preponderance of blasts in the peripheral blood or bone marrow (AML); however, they are closely associated through their shared lineage as well as their existence on a spectrum with some cases of MDS displaying increased blasts, a feature that reflects more AML-like behavior, and the propensity for MDS to transform into AML. It is increasingly recognized that the distinctions between these two entities result from the divergent patterns of genetic alterations that drive each of them. Mutations in genes related to chromatin-remodeling and the spliceosome are seen in both MDS and AML arising out of antecedent MDS, while mutations in genes related to signaling pathways such as  Myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML) are two disease entities that together form a spectrum of myeloid neoplasms with a common pathogenesis. The diseases are linked by similarities in their biology, clinical presentation, management, and through the capacity of MDS to transform into AML in the setting of clonal evolution or progression. The main risk factor for both diseases is advanced age, though they may also be predisposed by environmental exposures, and in a minority of cases, germline mutations –3. HowevThe notion that leukemia could be preceded by a pre-leukemic bone marrow failure state characterized by cytopenias and abnormal morphology began coalescing in the mid-1900s, nearly 100 years after acute leukemia was first described , 5. EvenA key feature that distinguished MDS from other bone marrow failure states was the observation in the 1980s that, like AML, MDS is a clonal process . This inUnderstanding the connection between MDS and AML hinges on an understanding of their shared pathogenesis. MDS and AML both arise through a process of clonal evolution in which the sequential acquisition of selectively advantageous mutations leads to clonal dominance, and eventually, malignant behavior. These mutations result in clonal hematopoiesis of indeterminate potential (CHIP), in which the mutated clone predominates over unmutated clones but doesn’t produce ineffective hematopoiesis or overt neoplasia as seen in MDS and AML –17. AlthCHIP is associated with an increased risk for progression to a hematologic malignancy, though only a minority of patients will develop overt malignancy . Some CHExpansion or evolution of the hematopoietic clone in CHIP or CCUS then leads to a progressively increased risk of developing a myeloid malignancy. The complex interplay of the underlying genetic and epigenetic changes determines the phenotype of the disease with respect to the MDS-AML spectrum. Additional genetic changes may drive further clonal and phenotypic evolution, resulting in the transformation of one disease phenotype (i.e. MDS) to another (i.e. AML) Figure 1DNMT3A, TET2, IDH1/2 are involved in the coordination of DNA methylation; ASXL1, BCOR, and EZH2 are involved in histone modification; SF3B1, SRSF2, ZRSR2, and U2AF1 encode components of the spliceosome; STAG2, SMC3, SMC1A, and RAD21 form components of the cohesin complex; RUNX1, GATA2, and ETV6 are transcription factors; TP53 is a tumor suppressor gene; and JAK2, NRAS, KRAS, FLT3, WT1, NF1, CBL, and PTPN11 are all involved in cell signaling. With some exceptions, mutations infrequently occur in multiple genes of the same category within the same clone. This is exemplified by the rarity of mutations in multiple spliceosome genes . Conversely, mutations in genes from multiple categories are often required for malignant transformation  mechanisms generates a highly inflammatory bone marrow microenvironment, which stifles normal hematopoiesis and drives further clonal evolution , 32. ThuThe biological and phenotypic differences between MDS and AML are underpinned by genetic differences.de novo AML), and AML with antecedent myeloid neoplasm   , 39. WhiL, sAML) . These Msyndrome .SF3B1 is another example in which the specific mutation correlates closely with the presence of ring sideroblasts. SF3B1 is seen in approximately 80% of patients with MDS with ring sideroblasts (MDS-RS) and single lineage dysplasia and 40% of those with MDS-RS with multilineage dysplasia  correlates with the burden of ring sideroblasts in the marrow. It is not entirely clear how SF3B1 mutation produces the RS phenotype (q13), isochromosome 17q, 17p deletion or loss of 17p, monosomy 13 or 13q deletion, 12p deletion or loss of 12p, 11q deletion, monosomy 7, 7q deletion, or loss of 7q, complex karyotype, and pathologic mutations in ASXL1, BCOR, EZH2, SRSF2, STAG2, U2AF1, and ZRSR2 . While some of the mutations in the spliceosome genes are hotspot mutations that may alter function, many of the MDS-associated mutations lead to loss of function and are therefore less amenable to drug targeting.While deletion 5q and nd ZRSR2 , 12. ComDe novo AML, on the other hand, frequently bears mutations in genes such as FLT3, NPM1, IDH1/2, CEBPA, WT1, PTPN11, and KRAS , thus leading to a proliferative phenotype  group. AML was defined by the presence of ≥30% blasts, whereas MDS was defined according to the percentage of blasts, the presence of ring sideroblasts, and the presence of monocytes . The catDespite the reality that the FAB system is now mostly considered in an historical fashion, it did highlight two important features of the natural history of MDS that remain relevant today: first, that cases with higher percentages of blasts were more likely to progress and transform into overt AML, an association that was captured by other prognostic scoring systems ; second,The advent of next generation sequencing has led to an ongoing process of redefining the borders of MDS, AML, and the subtypes within each of them. RUNX1::RUNX1T1, CBF::MYH11, PML::RARA alone was sufficient to make a diagnosis of AML , regardless of the blast percentage. In the most recent 2022 edition, this exception now extends to all AML subtypes with recurrent genetic abnormalities including AML with t/MLLT3::KMT2Ac, t/DEK::NUP214, inv(3)(q21.3q26.2) or t/GATA2, MECOM(EVI1), other rare recurring translocations, and mutated NPM1. While the ICC still requires a blast percentage of ≥ 10% in the setting of a genetically-defining lesion, the WHO does not include a lower limit in its definition of genetically-defined AML.Genetic features increasingly supersede blast percentage in the distinction and classification of MDS and AML as well. In the 2008 WHO classification, the presence of de novo AML from secondary AML. AML is now either classified as myelodysplasia related on the basis of specific genetic abnormalities or may be amended by the diagnostic qualifiers “progression from MDS” or “progression from MDS/MPN.”Similarly, the finding of dysplasia has lost some significance in the most recent ICC system. Historically, MDS required the presence of dysplasia in at least 10% of a single lineage; cases of cytopenias and clonal genetic abnormalities without dysplasia have more recently been classified as CCUS. The most recent ICC classification system, however, allows for the diagnosis of MDS, even without a finding of dysplasia if 5q deletion, monosomy 7/7q deletion, complex karyotype, or multi-hit TP53 are found. Furthermore, the finding of multilineage dysplasia is no longer used to distinguish The emphasis of genetics over morphology is particularly notable in the ICC classification of TP53-mutated myeloid neoplasms, which are now grouped together and set apart from other MDS and AML subtypes in order to convey the uniquely aggressive nature of these diseases, regardless of the blast percentage or presence of dysplasia.SF3B1 rarely presents at diagnosis with excess blasts, whereas MDS with AML-like mutations  almost exclusively presented with excess blasts  were more similar to patients with MDS (<20% blasts) in terms of their clinicopathology, molecular characteristics, and outcomes than they were to patients with ≥ 30% blasts . On the st count .While the use of a rigid threshold to define MDS and AML seems to compromise the nuanced conception of MDS and AML as representing sides of a spectrum of disease, it also has practical benefits. A blast threshold is used for trial enrollment to ensure a trial is enrolling the target population; it is helpful in providing guidance to community practitioners when deciding between the use of an MDS vs. AML treatment paradigm; and as a means for providing clinical annotation for translational research efforts. To balance the need for nuance and discrete disease categories, the term MDS/AML has been adopted by the European Leukemia Network and the International Consensus Classification to denote myeloid neoplasms with 10-19% peripheral or bone marrow blasts , 60. A wAn understanding of clonal evolution and the genetics of MDS and AML is critical for informed clinical decision making at the bedside. Clinical treatment paradigms have been designed to fit an either-or binary, but now must increasingly be adapted to a more nuanced biology-driven understanding of these diseases. Expectation management, deciding whether to pursue an MDS or an AML-oriented treatment paradigm, choosing the most appropriate AML therapy within an AML paradigm, and the decision of whether or not to pursue transplant all hinge on an understanding of a myeloid neoplasm’s past and future clonal trajectories. An extensive baseline characterization of the disease is of course a prerequisite in developing an accurate conception of the disease biology and behavior, but iterative reassessment over time is also crucial to the process of defining the disease. In patients without overt AML, there isn’t necessarily a penalty to deferring treatment , so provRUNX1::RUNX1T1, CBF::MYH11, or mutated NPM1. These patients have demographic and disease characteristics similar to patients with de novo AML, and the disease behavior is also more akin to that of de novo AML. They are also responsive to AML-type therapy, regardless of blast percentage at presentation , NPM1, or IDH1/2 in the setting of MDS heralds transformation to AML , which is often referred to as 5q minus syndrome. This form of MDS has a distinctive clinical phenotype characterized by a predominant anemia, normal or even increased platelets, few blasts with a low risk of transformation to AML, and a high rate of responsiveness to lenalidomide . In mostde novo and secondary AMLs.As previously noted, the definition of secondary AML has come to rest largely on the genetic features of the disease. In the most recent WHO and ICC classification systems, sAML  is defined as AML with defining genetic abnormalities such as unbalanced loss of chromosomal material and mutations in genes involved in the spliceosome. In the WHO system, patients with AML and a known history of MDS or MDS/MPN are classified together with those patients with AML and MDS-related genetic abnormalities, while in the 2022 ICC classification system, a history of MDS or MDS/MPN is denoted as a diagnostic qualifier. In both the WHO and ICC, the finding of dysplasia in AML is no longer used to distinguish de novo AML is a clinically important task. sAML is well-known to be associated with lower rates of complete remission and worse overall survival than de novo AML  called CPX-351. In a phase 2 study, outcomes were most improved in the subset of patients with secondary and treatment-related AML , leadingAs previously noted, the bone marrow blast percentage cut off that distinguishes MDS from AML in cases without AML-defining genetic features remains at 20%. We can acknowledge the need for such a cutoff without being dogmatic. There are some cases in which a patient with properly categorized high risk MDS, particularly those with excess blasts, may benefit from AML-type therapy such as intensive chemotherapy or venetoclax-based therapy. Some centers routinely consider patients with MDS-EB2 eligible for AML-type therapy  and inteVenetoclax-based therapy, on the other hand, has demonstrated acceptable safety and efficacy in early phase trials of untreated higher risk MDS, albeit dose-reduced compared to AML dosing . There iTP53 loss-of-function mutations are common in both MDS and AML, but their clinical significance depends largely upon whether the loss of function is monoallelic, biallelic (BiTP53), or is accompanied by a complex karyotype (CK). Biallelic loss of function may result from the acquisition of multiple point mutations or a single point mutation on one allele combined with loss of genetic material from 17p on the other allele resulting in copy neutral loss of heterozygosity. Patients with either MDS or AML with BiTP53 or CK-TP53mut have similarly dismal outcomes. In contrast, patients with monoallelic TP53 mutation without CK have outcomes that are intermediate between that of BiTP53/CK-TP53mut and Non-CK-TP53wt  interaction and agents engineered to restore the function of mutated TP53 proteins. CD47 is an immune checkpoint cell surface marker that is upregulated in high risk MDS and AML myeloblasts that binds SIRPα on circulating immune cells including macrophages, sending a “don’t eat me” signal that inhibits phagocytosis. Blocking this interaction through anti-CD47 (magrolimab) or SIRPα antibodies has shown promising efficacy in early phase studies, particularly in TP53-mutated myeloid neoplasms . APR-246Greater emphasis on drug development that targets the specific genetic drivers of disease will necessitate further relaxation of the boundaries that distinguish MDS from AML , 105. NoMDS and AML are inextricably intertwined through their shared pathogenesis, overlapping clinical features, and MDS’s predilection for transformation. Appropriate treatment of these entities requires an expertise with the biological characteristics of each and how these characteristics shape the treatment paradigms that come to be associated with each of them.AA and AD were responsible for the conception and writing of this manuscript. All authors contributed to the article and approved the submitted version.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.


AP duration was unaltered in TnT-R173W iPSC-CM. Nevertheless, TnT-R173W iPSC-CM showed a strikingly low stimulation threshold for AP- and [Ca2+]i-alternans. Myofilaments are known to play a role as intracellular Ca2+ buffers and here we show increased Ca2+ affinity of intracellular buffers in TnT-R173W cells, indicating increased myofilament sensitivity to Ca2+. Similarly, EMD57033, a myofilament Ca2+ sensitiser, replicated the abnormal [Ca2+]i dynamics observed in TnT-R173W samples and lowered the threshold for alternans development. In contrast, application of a Ca2+ desensitiser (blebbistatin) to TnT-R173W iPSC-CM was able to phenotypically rescue Ca2+ dynamics, normalising Ca2+ transient profile and minimising the occurrence of Ca2+ alternans at physiological frequencies. This finding suggests that increased Ca2+ buffering likely plays a major arrhythmogenic role in patients with DCM, specifically in those with mutations in cardiac troponin T. In addition, we propose that modulation of myofilament Ca2+ sensitivity could be an effective anti-arrhythmic target for pharmacological management of this disease.Dilated cardiomyopathy (DCM) is a major risk factor for heart failure and is associated with the development of life-threatening cardiac arrhythmias. Using a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model harbouring a mutation in cardiac troponin T (R173W), we aim to examine the cellular basis of arrhythmogenesis in DCM patients with this mutation. iPSC from control (Ctrl) and DCM-TnT-R173W donors from the same family were differentiated into iPSC-CM and analysed through optical action potential (AP) recordings, simultaneous measurement of cytosolic calcium concentration ([CaThe online version contains supplementary material available at 10.1007/s00395-022-00912-z. Dilated cardiomyopathy (DCM) represents the most common cardiomyopathy and is a major contributor to heart failure and sudden cardiac death . Roughly2+) handling and reduced contractility are key features of patient-specific iPSC-CM carrying TnT-R173W i), Fluo-3 was excited at 488 nm and emitted light (> 520 nm) converted to [Ca2+]i, assumingkd is the dissociation constant of Fluo-3 (864 nM), F is the Fluo-3 fluorescence; Fmax is the Ca2+-saturated fluorescence obtained at the end of each experiment , as determined during the decay of the caffeine-induced Ca2+ transient i at which buffers are half saturated, was significantly lower in DCM-TnT-R173W, compared to Ctrl , allowing estimation of the time course of changes of total cytosolic Ca2+ during ICa,L-triggered Ca2+ transient  as a function of the free cytosolic Ca2+ level  plotted against the free [Ca2+]i with unaltered slope in DCM-TnT-R173W. The unaltered slope shows that the slowed decay of the systolic free Ca2+ transient in DCM-TnT-R173W  showed no difference between groups, confirming unaltered Ca2+-dependence of NCX function. Since [Ca2+]i dependence of decay rate of total Ca2+  was unaltered during systolic and caffeine-induced Ca2+ transients, it can be concluded that [Ca2+]i dependence of SERCA activity is unaltered in DCM-TnT-R173W, which has been estimated based on the difference between the two respective slopes  was unaltered  and time constant of decay was greater in EMD57033-treated iPSC-CM, compared to Ctrl  i separately from alterations in the Ca2+ binding affinity of myofilaments. The latter represent the major Ca2+ buffers in cardiac myocytes and alterations in binding affinity are thought to have relevant impact on cellular Ca2+ homeostasis i as opposed to direct activation by ICa,L channels [2+ handling and contractile function [In the present study, we used iPSC-CM from DCM patients harbouring the TnT-R173 mutation. iPSC-CM represent myocytes at an immature developmental stage. iPSC-CM exhibit poor co-localisation between Iand RYR2 , resultichannels , 78. Nevfunction , 23. In function , 13, makfunction , 44, 77.Ca,L in DCM-TnT-R173W iPSC-CM. In contrast, SR Ca2+ content was unchanged in DCM-TnT-R173W iPSC-CM . In addition, Ca2+ removal by forward mode NCX was increased in DCM-TnT-R173W iPSC-CM to compensate for the higher Ca2+ influx through upregulated ICa,L. The mechanisms underlying increased ICa,L are beyond the scope of the present study. Nevertheless, unaltered mRNA expression of the underlying Ca2+ channel subunit, Cav1.2, renders intrinsic differences of its expression levels between DCM-TnT-R173W and Ctrl iPSC-CM unlikely . Further analysis of the biphasic ICa,L inactivation revealed unaltered time course of fast ICa,L decay , which is thought to be mainly due to Ca2+ dependent inactivation of ICa,L [Ca,L in DCM-TnT-R173W is unlikely due to reduced Ca2+-dependent inhibition of ICa,L in response to reduced free cytosolic Ca2+ levels. In accordance, increased cytosolic buffering due to EMD57033 leads to a similar decrease in Ca2+ transient amplitude but had no effect on ICa,L. ICa,L is regulated by various post-transcriptional and post-translational mechanisms including miRNA-dependent inhibition, phosphorylation and expression of accessory units [Ca,L alterations in DCM patients in general, but also those harbouring the TnT-R173W mutation.Our data show significant upregulation of I+ efflux , 67. Accry units , 56. FutCa,L, the triggered Ca2+ transient amplitude was smaller in DCM-TnT-R173W compared to Ctrl. Based on our experiments, we conclude that this is largely due to increased cytosolic Ca2+ buffering. In accordance, the total amount of Ca2+ released from the SR during ICa,L-triggered Ca2+ transients was unaltered in DCM-TnT-R173W . Therefore, impaired interaction between L-type Ca2+ channel and RyR2 may additionally contribute to reduced free ICa,L-triggered Ca2+ transient amplitude.Despite greater I2+ buffering, we employed a method that allows investigation of Ca2+ homeostasis in intact cardiomyocytes [2+ has been quantified by the fluorescent Ca2+ indicator Fluo-3, which represents an intracellular Ca2+ buffer itself. We assume that the contribution of Fluo-3 to intracellular Ca2+ buffering is comparable between experimental groups and does not contribute to the differences observed in the present study. Based on the dissociation constant (Kd = 0.864 µM) and an estimated buffer concentration (bmax) of 100 µM, the contribution of Fluo-3 to the calculated buffer curves is illustrated in Supplementary Fig. 5. In addition, the employed techniques do only allow indirect conclusions on altered Ca2+-binding to the troponin complex. Direct quantification of Ca2+ binding to troponin C will require further biochemical analysis that are beyond the scope of the present study [To quantify cytosolic Camyocytes . Intracent study .2+ handling abnormalities in iPSC-CM carrying a specific mutation in cardiac TnT that has been associated with the occurrence of DCM. Given the multifactorial aetiology of DCM, it is unclear whether reduced Ca2+ uptake by SERCA, secondary to increased Ca2+ buffering by myofilaments may represent a “final common pathway” underlying arrhythmogenesis in DCM patients [2+ reuptake into the SR [2+ sensitivity of contraction has also been found in patients and a dog model of pacing induced DCM [2+ sensitivity were abrogated after treatment with the catalytic subunit of PKA, suggesting that, as in DCM caused by TnT-R173W mutations, the increased Ca2+ sensitivity of myofilaments may be due to a reduction in PKA-mediated phosphorylation of myofibrillar regulatory proteins.In the present study, we investigated Capatients . Nevertho the SR . Furtheruced DCM , 75. The2+ may contribute to arrhythmogenesis in various cardiac diseases, modulation of myofilament Ca2+ sensitivity may represent an important novel concept to prevent cardiac arrhythmias [Based on our findings and given the fact that increased myofilament affinity for Cahythmias , 59.2+ binding of myofilaments is a classical therapeutic concept to improve contractile dysfunction in heart failure patients. Levosimendan and omecamtiv represent traditional drugs aiming to improve contractile force by increasing Ca2+ sensitivity and Ca2+-myofilament binding [2+ [Targeting Ca binding , 49. Nevding [2+ , 20.2+ alternans in mouse hearts harbouring Ca2+-sensitising TnT mutations in vitro [2+ alternans in DCM-TnT-R173W iPSC-CM. Similar to blebbistatin, mavacamten, a small molecule modulator of β-cardiac myosin, which has been recently evaluated in patients with hypertrophic cardiomyopathy, has also been shown to reduce Ca2+ affinity of myofilaments [Blebbistatin is an inhibitor of the myosin ATPase and has been shown to prevent the occurrence of Cain vitro . Accordiilaments . Both blSupplementary file1 (DOCX 2314 KB)Below is the link to the electronic supplementary material.


RyR2 protein has multiple functional domains with specific roles, and four of these RyR2 protomers are required to form the quaternary structure that comprises the functional channel. Numerous mutations in the gene encoding RyR2 protein have been identified and many are linked to a wide spectrum of arrhythmic heart disease. Gain of function mutations (GoF) result in a hyperactive channel that causes excessive spontaneous SR Ca2+ release. This is the predominant cause of the inherited syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). Recently, rare hypoactive loss of function (LoF) mutations have been identified that produce atypical effects on cardiac Ca2+ handling that has been termed calcium release deficiency syndrome (CRDS). Aberrant Ca2+ release resulting from both GoF and LoF mutations can result in arrhythmias through the Na+/Ca2+ exchange mechanism. This mini-review discusses recent findings regarding the role of RyR2 domains and endogenous regulators that influence RyR2 gating normally and with GoF/LoF mutations. The arrhythmogenic consequences of GoF/LoF mutations will then be discussed at the macromolecular and cellular level.The ryanodine receptor (RyR2) has a critical role in controlling Ca Membrane depolarization causes a rapid rise in [Ca2+] from nanomolar to micromolar levels in the narrow dyadic cleft formed between T-tubule and SR junctional membrane, which increases RyR2 activity in a sigmoidal function of cytosolic Ca2+ concentration. RyR2 can be fully activated at ~100 μM Ca2+  critical for Ca2+ interaction  is increased, e.g., evoked by an action potential, but suppresses RyR2 opening at diastolic [Ca2+] [2+ spark amplitude was found to be fairly insensitive to cluster sizes greater than ~20 RyR2 (quantified indirectly using fluorescently-labeled FKBP12.6) [2+ flux rate in proportion to the number of open channels times the trans-SR [Ca2+] gradient, this will more rapidly deplete junctional SR [Ca2+], thus reducing the driving force for Ca2+ release and cause a gradual loss of regenerative capacity and finally Ca2+ spark termination [Early investigations using electron microscopy established that square-shape RyR2 tetramers (~30 × 30 nm) organize as discrete clusters in a quatrefoil crystalline arrangement on junctional SR membrane . Purifiec [Ca2+] . Ca2+ spKBP12.6) . Althougmination .2+ concentration decreases rapidly as it diffuses further from its source, therefore the probability of one cluster causing Ca2+-induced Ca2+ release (CICR) in distant clusters falls sharply with distance, and this limits uncontrolled activation. However, if two or more clusters are sufficiently close then the increase in cytosolic [Ca2+] can be enough to activate its neighbor, such that closely spaced RyR2 clusters (~100–150 nm edge-to-edge) are predicted to act together as functional Ca2+ release units (CRU) in the formation of Ca2+ sparks  at nearby clusters increases sufficiently to activate nearby CRU spaced ~0.5 µm apart in the same Z-line [2+ increases the interactions between CRU and the occurrence of propagating Ca2+ waves [2+ wave generation is predicted to be highly dependent on Z-disk spacing, by influencing the initial Ca2+ flux needed to raise Ca2+ above the threshold for CRU activation at nearby sites [1-adrenergic receptor blocker, metoprolol [2+ wave frequency and triggered activity, despite similar Ca2+ spark frequency between treated and untreated cells.An increase in Cae Z-line , or acro2+ waves . The likby sites . Shorterby sites  and righby sites . The shotoprolol , and thi2+ wave propagation by reducing the longitudinal inter-CRU distance. Rogue RyR2 occur infrequently in normal ventricular myocytes [2+ sparks occur almost exclusively from RyR2 clusters in Z-lines [2+ wave propagation in normal cardiomyocytes. There was no difference in the proportion of non-Z-line RyR2 in human persistent AF [2+ waves in a pathological context. Rogue RyR2 could be involved in substantial non-Ca2+ spark mediated leak, involving brief openings of small size clusters that would minimally raise cytosolic Ca2+ and would be below the optical limit of detection using fluorescent Ca2+ sensitive dyes [2+ spark-independent leak in failing rabbit ventricular myocytes contributed to reduced SR Ca2+ load [2+ release on SR Ca2+ load during normal excitation–contraction coupling.‘Rogue’ RyR2 located on the network SR outside of SR junctions could, in principle, facilitate Camyocytes  and Ca2+ Z-lines , suggeststent AF ; howeverstent AF , so it iive dyes . Increasa2+ load  and woul+/Ca2+ exchanger is one of the main Ca2+ removal pathways that restore cytosolic Ca2+ during diastolic Ca2+ waves back towards resting levels. Near resting membrane potentials, NCX works in the forward mode to remove 1 Ca2+ ion from the cytosol in return for 3 Na+ ions entering. This net electrogenic action produces a transient inward current that depolarizes the resting membrane potential, causing events called delayed afterdepolarizations (DADs). If DADs reach the threshold for Na+ channel activation, then triggered action potentials can occur and activate surrounding cells resulting in ectopic electrical activity in the heart that can degenerate into ventricular tachycardias that can lead to sudden cardiac death. DADs are believed to be the underlying event responsible for arrhythmia in CPVT [2+ waves in cells in the heart are unlikely to propagate the electrical activity further. Using optogenetic activation of channelrhodopsin-2 expressing cardiac-specific transgenic mice, it was estimated that ~2000 myocytes must depolarize near-simultaneously to initiate premature ventricular contractions [2+ regulation is deranged by factors causing RyR2 GoF phenotype and if there is coupling to entrain oscillations between cells. Indeed, Borile et al. found that during beta-adrenergic stimulation in ventricular heart slices from mice harboring the CPVT-causing RyR2-R2474S heterozygous missense mutation, cells surrounding those that exhibited DADs also had a greater probability of Ca2+ waves occurring almost synchronously [The Na in CPVT . Fortunaractions . The storonously .+ currents and inward currents  and are believed to be a major source of ventricular tachyarrhythmias [2+ release during repolarization increases inward INCX current and has been recognized as being involved in EADs for many years [NCX is a non-regenerative current  and was believed to contribute mainly by delaying repolarization and allowing time for reactivation of other inward currents (such as INa or ICa) to drive the upstroke of the EAD [2+ release, in the form of ‘late Ca2+ sparks’ [2+ transient decay triggered by ICa during the AP plateau and increased cytosolic Ca2+. The frequency of late Ca2+ sparks was greater in failing rabbit ventricular myocytes [2+ ripples’. Under conditions of increased Ca2+ load, late Ca2+ sparks-mediated INCX could take a dominant role in driving the depolarization [2+ release due to hyperactive RyR2 (increased Ca2+ sensitivity and reduced refractoriness) contributes to EAD genesis by increasing INCX [2+ sparks can fire more than once during a Ca2+ transient, after an apparent refractory period that is likely limited by the rate of junctional Ca2+ refilling [NCX at positive membrane potentials. The involvement of SR Ca2+ release in EADs is further apparent in the case of conditional SERCA2a knockout mouse ventricular myocytes [2+ store is essentially absent in knockout mice, demonstrated by the absence of Ca2+ sparks, meaning contraction is solely maintained by an increase in trans-sarcolemmal Ca2+ entry through ICa and a slight decrease in K+ current density. Despite such major electrophysiological adaptations, myocytes from SERCA2a knockout mice do not exhibit greater EAD incidence than control mice. It is worth noting that the major ion channels implicated in causing EADs are highly coupled to each other  occur during repolarization due to a mismatch between outward Khythmias . Similarny years   [2+ sparks had a faster rate of rising and shorter refractory period between consecutive Ca2+ sparks, possibly suggesting more RyR2 channels opening during each event due to increased Ca2+ sensitivity. The Ca2+ transient decay was slower and more late Ca2+ sparks occurred in CaMKII𝛿c over-expressing mice than phospholamban knockout controls, which could be caused by RyR2 Ca2+ hypersensitivity, but could also be due to CaMKII𝛿c facilitation of ICa [2+ transient and promote late Ca2+ sparks [2+ release in ventricular myocytes from mice with the CPVT-causing CSQ2-R33Q mutation that results in a GoF RyR2 phenotype, suggesting systolic Ca2+ handling could also be affected by GoF mutations [Factors that increase RyR2 Ca+ sparks . Post-trview see ). Guo et uptake) . They fon of ICa  that wou+ sparks  Liu et autations .+/− missense mutation [2+ transient were depressed and SR Ca2+ load was greater in RyR2-A4860G+/− ventricular myocytes compared to WT cells. Continued Ca2+ entry during the action potential via ICa with less efficient evoked SR Ca2+ release was proposed to increase net Ca2+ entry, overloading the SR, and resulting in frequent prolonged systolic Ca2+ release and EADs under basal conditions, and at greater frequency during stimulation with isoproterenol. They proposed that the extended Ca2+ release during EADs help unload the SR in this LoF model, although this will be balanced somewhat by the increased Ca2+ entry through ICa at depolarized membrane potentials. Alternatively, it was recently proposed that the reduced Ca2+ transient rate of rise in RyR2-A4860G+/− could cause less Ca2+-dependent inactivation and therefore prolong ICa during the action potential plateau, which would increase action potential duration both directly and indirectly (through increased INCX), although this remains to be demonstrated experimentally [+/− mutation also showed increased EADs and SR Ca2+ load in isolated myocytes and re-entrant arrhythmias following burst pacing in isolated hearts, although this occurred without a change in steady state Ca2+ transient amplitude or rate of rise, and a markedly increased ICa density [The rare LoF RyR2 mutations described in mutation . Under bmentally . Sun et  density . These i2+ release properties in the native environment using advanced imaging technologies offers an exciting future direction that promises to better our understanding of how cardiac excitation-contraction coupling is regulated in health and disease.The list of mutations in genes encoding RyR2 and its regulatory proteins associated with pro-arrhythmic phenotype continues to grow. These are revealing new insights into the structure/function relationship of RyR2 that is communicated both within and between subdomain interactions of this critical ion channel. It is now generally accepted that there is no common molecular defect, no single unifying hypothesis to explain how RyR2 GoF mutations result in typical CPVT. Moreover, molecular characterization of site-specific mutations may help explain the apparent diverging phenotypes in GoF and LoF RyR2 mutations. However, as LoF mutations have only fairly recently been the subject of attention, further work is needed to establish the extent to which their arrhythmogenic substrates are unique, or whether there is also some overlap with GoF mutations, as this may have important implications for the optimal therapeutic approach. Improved capabilities to investigate subcellular changes in RyR2 behavior and Ca


With its unique computer paradigm, the Ising annealing machine has become an emerging research direction. The Ising annealing system is highly effective at addressing combinatorial optimization (CO) problems that are difficult for conventional computers to tackle. However, Ising spins, which comprise the Ising system, are difficult to implement in high-performance physical circuits. We propose a novel type of Ising spin based on an electrically-controlled magnetic tunnel junction (MTJ). Electrical operation imparts true randomness, great stability, precise control, compact size, and easy integration to the MTJ-based spin. In addition, simulations demonstrate that the frequency of electrically-controlled stochastic Ising spin (E-spin) is 50 times that of the thermal disturbance MTJ-based spin (p-bit). To develop a large-scale Ising annealing system, up to 64 E-spins are implemented. Our Ising annealing system demonstrates factorization of integers up to  Combinatorial optimization problems have long been a tangled mess for conventional computers. With the scale of the problem increasing, the consumption of time and hardware resources increases exponentially. Heuristic search techniques, such as the simulated annealing algorithm, have been utilized to search for a solution within a limited time. However, their search effectiveness remains unsatisfactory. It is anticipated that quantum annealing ,2 would Ising annealing was offered as an alternative scenario between classical computing and quantum computing. Ising annealing is a novel non-von Neumann computing architecture that specializes in CO problems ,4,5,6. TThis paper proposes an electrically-controlled stochastic-MTJ-based Ising spin (E-spin) and an E-spin-based probabilistic Ising annealing algorithm (QFactor). The E-spin has good thermal stability, compact size, true randomness, and high controllability. In addition, the E-spin significantly achieves a 92.2% smaller area and 2% power consumption reduction compared to LFSR  based diThe spin-transfer torque magnetic tunnel junctions (STT-MTJs) possess the property of stochastic switching ,19,20, abability ,24.The construction of the Double-MTJ employed in this work is depicted in The process of operation is shown in The proposed E-spin was constructed based on the stochastic STT-MTJ, shown in The n-type metal–oxide–semiconductor (NMOS) transistor is a current source that supplies the electrically-controlled stochastic STT-MTJ with an appropriate amount of current. Depending on the direction and amount of current passing through it, the STT-MTJ may change its state (high or low resistance), altering the voltage of INP. Assuming that the high-resistance MTJ corresponds to The circuit’s current direction is depicted in The output of the For the spin dice, tion see  in our dThe temperature, which is determined by the surroundings and operating conditions, has a direct impact on the switching frequency for thermal disturbance MTJ (p-bit) . HoweverAccording to the spin operation modes mentioned in 15, it can operate at the frequency of 10 MHz for 3.17 years. The endurance can be increased even more if the SOT-MRAM is included [In the existing non-volatile memories, the endurance of MRAM is an obvious advantage compared to RRAM and PCRAM. The endurance of STT-MTJ with multiple CoFeB/MgO interfaces could theoretically reach up to onal MTJ . Construincluded . BecauseThe Ising system is composed of Ising spins and their interconnections. The Hamiltonian of the Ising system is:By mapping the task function into the Ising Hamiltonian and mapping the best answer to the lowest value of the acquired Ising Hamiltonian, the task can be transformed into searching for the lowest energy state of the Ising system. After the annealing procedure, the system energy will gradually descend and reach the low energy state. At this point, a workable solution is discovered.An illustration of the combinatorial optimization problem is the integer factorization problem. The integer factorization problem, which is still challenging for traditional computers, is now widely used in the RSA (Rivest–Shamir–Adleman) encryption technique.F is an According to , the IsiWe use E-spins to represent each bit of Y are discrete, H is a discontinuous function. Since the two variables The integer factorization problem differs from other combinational problems in that the former requires the optimal solution, whilst the latter just requires a good enough response. In other words, the output answer must meet higher standards because it will not be legitimate if the product of two factors does not exactly equal the number F.Making the non-continuous function into a continuous function is one of the effective ways to address discrete optimization problems since it speeds up the process of convergence to the local minimum. Therefore, in the second step, the discrete function In the third step, gradient descent was employed to reach the local minimum, as deep learning algorithms do in neural networks. Variable Fourthly, the continuous variable Lastly, verify that If Map If the calculated Accordingly, if Repeat step 2 to step 5 until the correct solution is found.The overall diagram of the proposed Ising annealing system is shown in The key to obtaining a better solution for an Ising annealing system is jumping out of the local optimal solution. Therefore, randomness is required in the annealing procedure to escape the local minimum. A significant difference between QFactor and other Ising annealing algorithms is the production of randomness. The QFactor algorithm fully utilizes the stochastic nature of E-spin, negating the need to use another randomness as the motivation of the annealing procedure.In addition, QFactor employs randomness differently than traditional heuristic search algorithms, such as simulated annealing, which selects a random number of Except for Ising annealing algorithms and heuristic search algorithms, there are other traditional methods to solve integer factorization problems, such as numerical calculation algorithms. The General Number Field Sieve (GNFS) method is the most effective numerical computation algorithm for solving the integer factorization problem at present. However, because of the operation’s complexity, hardware optimization is challenging. Nevertheless, GNFS is a specific algorithm for a single purpose. The QFactor algorithm provides an easy-to-implement way to solve more general discrete optimization problems.The schematic view of the CMOS-based stochastic Ising spin is shown in Since the LSB of the two factors For instance, the averaged number of cycles needed to factor 32-bit integers using the simulated annealing, Ising annealing, and trial division methods, respectively, is As the integer bit width . The thermal disturbance MTJ-based spin (p-bit) utilizes thermal-controlled unstable MTJ to realize a true-random-number-generator (TRNG).The MTJ is simulated by a Verilog-A model with 7.425 KΩ high resistance and 2.75 KΩ low resistance. The Verilog-A model is used to simulate the behavior of MTJ under various voltages, which is shown in The current-time simulation of the E-spin is shown in data see  are calcThe primary shortage of LFSR is that it is not a truly random source. The random number generated by an n-bit LFSR will loop for a maximum of The primary defect of thermal disturbance MTJ is the intricate voltage offset operation requirement for each bit, which restricts the application in large-scale issues.The proposed E-spin outperforms the p-bits in stability, speed, and large-scale integration. Firstly, the E-spin adapts the electrical operation, which helps in precise control. Meanwhile, the p-bit uses the thermal-disturbance-controlled MTJ, the frequency of which is influenced by the operating temperature. As a result, the frequency of p-bit is unpredictable, which could cause problems if interconnected with other modules. Additionally, the p-bit is in demand of the voltage offset for each bit. As a result, building an array out of p-bits is challenging.Secondly, the electrical operation also permits the E-spin to operate with a high frequency. The minimum pulse width of the read/write operation is 2 ns. The frequency of the E-spin could reach higher than 50 MHz. In comparison, the frequency of p-bit is less than 1 MHz. The working frequency of the E-spin is considerably higher.Lastly, the E-spin could easily integrate with logic circuits. We demonstrate the fabrication of 64 E-spins with logic circuits under the 40 nm technology node, which shows great potential in large-scale integration. Nevertheless, ref  only demThis paper proposes a stochastic Ising spin (E-spin) based on the electrically-controlled STT-MTJ. The probabilistic Ising annealing algorithm (QFactor) is based on the E-spins. The E-spin has true randomness, excellent stability, fine control, high frequency, compact size, and is simple to integrate thanks to its electrical operation. Using the gradient descent algorithm and E-spins to escape the local minimum is a novel approach to solving optimization problems. The E-spin achieved 50 times the frequency of p-bit. Up to 64 E-spins are implemented to construct a large-scale Ising annealing system. Factorization of integers up to


The group I adenylyl cyclase  isoforms, including ADCY1, 3, and 8 , are stimulated by Ca2+ and thus functionally positioned to integrate cAMP and Ca2+ signaling. Emerging lines of evidence have suggested the association of the Ca2+-stimulated ADCYs with bipolar disorder, schizophrenia, major depressive disorder, post-traumatic stress disorder, and autism. In this review, we discuss the molecular and cellular features as well as the physiological functions of ADCY1, 3, and 8. We further discuss the recent therapeutic development to target the Ca2+-stimulated ADCYs for potential treatments of psychiatric and neurodevelopmental disorders.As the main secondary messengers, cyclic AMP (cAMP) and Ca Adenylyl cyclase (ADCY) activity accounts for the basal level as well as activity-dependent production of cAMP, which is a main second messenger and regulates a wide spectrum of intracellular signaling molecules . In the s and Ca2+ and inhibited by Gi. The group II ADCYs include ADCY2, 4, and 7. They are stimulated by Gs and not responsive to Ca2+ and Gi. The group III ADCYs include ADCY5 and 6. They are stimulated by Gs and inhibited by Ca2+ and Gi. The group IV ADCY includes ADCY9, which is only stimulated by Gs and, unlike other ADCYs, not responsive to the general ADCY activator forskolin. The group V ADCY includes ADCY10, which is a structurally distinct soluble cytoplasmic protein whose activity is regulated by Ca2+ and bicarbonate but not GPCRs  are membrane-bound proteins and share similar structures with a short N-terminal cytoplasmic domain (the C1 domain) two transmembrane domains  connected by the C1 cytoplasmic loop, and the C2 cytoplasmic domain at the C-terminus  Figure . Throughot GPCRs . ADCY10  neurons . Human g2+ -binding domain in the C1 or C2 region of some ADCYs suggests that certain isoforms can be regulated by Ca2+ . Affinitng ADCYs . Among and ADCY8 . As cAMPignaling .2+  can cause ∼2-fold increase of ADCY3 activity in vitro (2+ (at ∼100–300 nM) significantly inhibits hormone- and Gαs-stimulated ADCY3 in vivo , which is triggered by Ca2+ depletion from the internal store, may lead to more robust Ca2+ increase and stimulate ADCY8. In the non-neuronal HEK293 cells, when ADCY8 and the CCE functional molecules STIM1 and Orai1 are over-expressed, they colocalize in lipid raft domains of the cell membrane. Functionally, while Ca2+ store depletion alone fails to activate ADCY8, over-expression of STIM1 and Orai1 along with Ca2+ store depletion leads to ADCY8 activation   . Howevereceptors . Furthereceptors . Thus, tnd ADCY8 . Regardi2+-stimulated ADCYs, cAMP and Ca2+ signaling may converge and tune specific signaling networks and, in turn, regulate cellular functions relevant to neurotransmitter receptor activity, gene transcription, and translation /ERK1/2 -MSK1 (mitogen- and stress-activated protein kinase-1)-CREB (cAMP responsive element binding protein), the Ca2+/cAMP-ERK1/2-eIF4E (eukaryotic translation initiation factor 4E)/4EBP1 (Eukaryotic translation initiation factor 4E-binding protein 1)/S6K1 (ribosome protein S6 kinase 1), and the Ca2+/cAMP-PKA/CaMKII-GluR1 (glutamate ionotropic AMPA type subunit 1) cascades  and Adcy8 KO mice, respectively  mice -mediated transcription of bdnf (brain-derived neurotrophic factor)  mice . The Adcpocampus , suggestpocampus . Consist factor) , mice ladnf mRNA . Converspocampus . Overexpl cortex .1/2 may impinge on ribosome activity and, in turn, regulate protein synthesis. In vitro and in vivo studies demonstrate that inhibition of ERK1/2 leads to impairments of activity-dependent upregulation of eIF4E, 4EBP1, and S6K1 phosphorylation  at S845, AMPAR-mediated EPSCs, and surface GluR1 are decreased at the thalamocortical synapses  of GluR1 are targets of PKA and CaMKII/PKC (protein kinase C), respectively , and funsynapses .Adcy1, detected by Northern blot and in situ hybridization, is predominantly expressed in the nervous system tissues including brain, adrenal gland, and retina . Notably, Adcy1 mRNA level is overwhelmingly higher in excitatory neurons than inhibitory neurons in the hippocampus but not in other regions , Western blot with a validated antibody -ADCY1 is expressed in both dendrite and axon. The punctate and discrete HA-ADCY1 colocalizes with synaptophysin and synapsin I in cerebellar neurons, indicating enrichment in the synaptic compartment . WesternAdcy1 mRNA in cortex, hippocampus, and cerebellum, studies with the Adcy1 KO mice have found that activity-dependent potentiation of synaptic efficacy in these brain regions requires ADCY1  channels causes the influx of Na+ and Ca2+, leading to sensory neuron activation and the subsequent olfactory detection process. Deficiency of ADCY3, which is the only ADCY in olfactory cilia, causes anosmia   .In brain neurons, ADCY3 is localized in the primary cilium , which iAdcy8 is expressed in both the CNS and the peripheral non-neuronal tissues  and postsynaptic marker  proteins . The end KO mice . The pos KO mice .2+/cAMP-mediated signaling has been detected as molecular outcomes that are associated with various aspects of psychiatric and neurodevelopment disorders. Within the Ca2+/cAMP signaling network  gene and deficient expression of its gene product FMRP (FMR1 protein). FXS is a neurodevelopment disorder and leading cause of intellectual disability and autism  mice (1/2/PI3K (phosphoinositide 3-kinases)-S6K1 signaling in FXS  , which id autism . Among vd autism , the RNAd autism . Along wd autism . Mining KO) mice . The enhg in FXS . The caudeficits . Interesdeficits .Adcy1 gene, as suggested by the human genome-wide association study (GWAS), is a potential genetic risk factor for schizophrenia  identifies an association of schizophrenia genes with reduced LTP, abnormal spatial learning, and neurodevelopment , it is feasible to detect whether the lithium effects are attenuated or potentiated.Alterations of disorder . Althougdisorder . BipolarAdcy3 polymorphism with depression  sleep, hypo-locomotion, neophobia, higher immobility in the tail suspension and forced swimming test, impaired nesting behavior and impaired sociability  on a chromosome region that is homologous to human 8q24, increased Adcy8 mRNA level is detected in ventral hypothalamus and piriform cortex and associated with avoidance behavior   . In contlus maze . The Adcing test .Adcy8 in hypothalamus suggests potential functions in regulating stress responses through the HPA  axis. A GWAS study implicates a suggestive association of Adcy8 with posttraumatic stress disorder  along with more risk-taking rather than anxiogenic behavior (The high expression level of disorder . Adcy8 dne level . Intrigubehavior .2+-stimulated ADCYs, it is necessary to develop pharmacological reagents. To date, several small molecule inhibitors against ADCY1 have been developed and tested in certain animal models. Drugs showing specific regulatory activity for ADCY3 and ADCY8 have not been reported yet.To achieve the therapeutic potential of targeting the Ca1/2/Akt-S6K1 signaling and rescues repetitive behaviors and social deficits (By using a structure-based rational design compound library, deficits .1/2/Akt activity and protein synthesis in the Fmr1 KO neurons. Peripheral administration of carbamazepine corrects hyperlocomotion and social deficits and improves learning and memory in the Fmr1 KO mice (Although NB001 is currently tested for safety in human clinical trials , it is n KO mice .Another ADCY1 inhibitor has been identified by screening a natural product derivatives library. ST034307 shows an IC50 of 2.3 μM against ADCY1 and no detectable inhibition against other ADCYs . InteresAs the backbone structures of the adenosine-based NB001 and chromone-based ST034307 predict limitation and drawback of these compounds as practical therapeutic reagents, recent effort aims to identify ADCY1 inhibitors with different structures. The oxadiazole-based AC10065 at micromolar concentrations can suppress both ADCY1 and ADCY8 . New dru2+-stimulated ADCYs in regulating various aspects of neuronal property and behavior. It is evident that a distinct ADCY isoform, rather than general and overall cAMP level, may specifically control an isoform-specific cellular and physiological function. Elucidation of Ca2+-stimulated ADCY function in distinct brain regions and distinct cell types may help to develop precise intervention of the disorder-specific pathology. Development of high affinity isoform-specific drugs with favorable pharmacokinetics and toxicity profile will lead to practical intervention to promote mental health and alleviate symptoms associated with certain psychiatric and neurodevelopment disorders. Further, interpretation with the current animal models should consider non-specific effects of global gene deficiency, complication of different genetic backgrounds and validity of the behavior outcome. Precision preclinical models with more direct face and construct validity need to be developed.In summary, previous studies have demonstrated the function of Ca


The sarcoplasmic reticulum contains several proteins, some of which support Ca2+ storage and release, while others regulate the formation and maintenance of this highly convoluted organelle and mediate the interaction with other components of the muscle fiber. In this review, some of the main issues concerning the biology of the sarcoplasmic reticulum will be described and discussed; particular attention will be addressed to the structure and function of the two domains of the sarcoplasmic reticulum supporting the excitation–contraction coupling and Ca2+-uptake mechanisms.The sarcoplasmic reticulum of skeletal muscle cells is a highly ordered structure consisting of an intricate network of tubules and cisternae specialized for regulating Ca 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 [2+ 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) . 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 sarcoplasmic reticulum (SR) is a specialized form of the endoplasmic reticulum of muscle cells, dedicated to calcium ion (Cayofibril . These eles (TT) . The strThe proper relationship between the elongated tubules of the l-SR and myofibrils is a pivotal structural prerequisite to organize the different domains of the SR and allow an efficient muscle contraction ,2. As of2+ 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 [The triad is the membrane structure that supports ECC in skeletal muscle fibers by allowing the interaction between the dihydropyridine receptor (DHPR) and RyR1 . The DHPtraction .The events that characterize TT maturation and triads formation have been morphologically defined by electron and confocal microscopy studies. These revealed that the juxtaposition of j-SR and TT begins in the embryonic (E) period of development and evolves through sequential steps in the postnatal life ,15. In mTT development appears to start immediately after that of the SR. At day E15, small tubular invaginations can be observed at the periphery of developing muscle fibers. By day E19, these invaginations expand deeper into the fiber, although remaining almost longitudinally oriented. After birth, the TT system starts acquiring its final transverse position over the borders between the A and I bands, achieving its definitive localization at around three weeks of age in mice ,14. A reWhile several details of the morphological events that characterize TT and SR maturation have been defined, the molecular mechanisms that drive this process remain more elusive. RyR1 and DHPR, although essential for ECC, have been shown to be dispensable for the formation of triads; indeed, triad formation was observed in knockout mice for RyR1, also called “dyspedic” mice since RyRs were defined as “junctional feet”. Triad formation was also observed in mice carrying muscular dysgenesis in DHPR, a natural mutation that causes lethal paralysis in mice. Finally, mice lacking both RyR1 and DHPR also show triad formation, further supporting the idea that these two proteins are not directly involved in triad formation or maintenance ,20,21. SAmphiphysin 2, also known as Bridging integrator-1 (Bin1), is a member of a family of ubiquitous proteins able to shape lipid membranes; thanks to their BAR (Bin/Amphiphysin/Rvs) domain, they can interact with membrane lipids and detect and/or generate membrane curvatures . Bin1 caCaveolin-3 (Cav-3) is the muscle-specific isoform of caveolins, which mediates the formation of peripheral invaginations in cholesterol-rich regions of the plasma membrane ,34,35. C2+ [DYSF gene cause myopathies, including limb–girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM), and distal anterior compartment myopathy [2+-dependent vesicle fusion with the plasma membrane [2+-activated chloride channel, which was also suggested to display phospholipid scramblase activity [2+ overload due to membrane damage [Dystrophy-associated fer-1-like protein (DYSF) is an integral protein belonging to the ferlin family, characterized by multiple C2 domains able to bind membrane phospholipids in the presence of Ca2+ . It is m2+ ,49. Mutamyopathy ,51,52. Dmembrane . Annexinactivity . Mutatioactivity ,56. Moree damage .Mitsugumin-53 (MG53), also known as tripartite motif 72 (TRIM72), is a skeletal and cardiac muscle-specific protein that contributes to vesicle trafficking involved in the membrane repair machinery ,58. MG532+ transients during skeletal muscle contraction [Mitsugumin-29 (MG29), which belongs to the synaptophysin family and is characterized by early expression during myogenesis, associates with newly formed SR vesicles ,62. Duritraction .MTM1 are associated with X-linked myotubular myopathy, a skeletal muscle disorder that results in severe muscle weakness with abnormal nuclei positioning [SPEG have been linked to centronuclear myopathies with disruptions of triadic structures and impairment of ECC [Myotubularin (MTM1) is a lipid phosphatase localized at triads. Mutations in human itioning . Mice knitioning . MTM1 deitioning . This suitioning . On the itioning , indicatitioning ,73. Finaitioning ; mutatiot of ECC .Junctophilins mediate the apposition between ER/SR and plasma membrane in different cell types. In mammals, Junctophilin 1 (JPH1) and Junctophilin 2 (JPH2) are expressed in striated muscles ,77,78. A2+ release complex [2+ homeostasis due to a partial loss in RyR1 and DHPR co-localization [2+ homeostasis [In skeletal muscle, both JPH1 and JPH2 are present at triads, where they are essential for the formation and maintenance of these contact sites. In addition to their structural role, JPHs can also bind and regulate various proteins at triads, acting as a scaffold for assembly of the Ca complex ,82,83. J complex ,89,90,91 complex . Downreglization ,85,86. Jlization ,94, whillization . In the lization ,99,100. lization . The seclization ; severaleostasis ,104,105.eostasis ,104. Neveostasis ,104,105.2+ binding proteins calsequestrin (CASQ) and histidine-rich calcium (HRC) binding protein [At triads, several proteins participate in the ECC process, including integral membrane proteins such as RyR1, DHPR, triadin, junctin, j-SR protein 1 (jp45), and mitsugumin-56, the STAC3 adaptor protein, or luminal proteins such as the Ca protein ,106,107 2+ release channels that represent the largest ion channels known to date. In skeletal muscle, two RyR isoforms, RyR1 and RyR3, are expressed, although only RyR1 is essential for ECC activation. In skeletal muscle, RyR1 is primarily activated by a conformational coupling with the DHPR, a mechanism defined as depolarization-induced Ca2+ release (DICR) [2+-induced Ca2+-release (CICR) mechanism, whereby an increase in cytosolic Ca2+ causes RyR3 opening and, thus, Ca2+ release from intracellular stores [2+ concentration, or, in the micromolar range, as a channel inhibitor [2+, Homer 1c, the scorpion venom imperatoxin, halothane, dantrolene, PCB95, ryanodine, ruthenium red, methylxanthines, and 4-chloro-m-cresol [RYR1 that result in RyR1 hyperactivation, leading to massive and uncontrolled Ca2+ efflux from the SR. Dantrolene was historically used as a skeletal muscle relaxant, and later, it was demonstrated to be able to block calcium release from the SR. Although the molecular mechanism responsible for dantrolene action on RYR1 is still not completely defined, its introduction for the clinical treatment of MH led to a significant decrease in mortality, from more than 90% to less than 5% [2+/CaM-dependent protein kinase II (CaMKII), and phosphatases  [RyRs are a family of Cae (DICR) . At varir stores ,108,109.r stores . RyRs har stores ,111. EM r stores ,111,112.nhibitor . One of nhibitor . RyR1 isnhibitor ,116. Addm-cresol ,118. Amo than 5% . Finally PDE4D3) .2+ channels located on the TT. The skeletal muscle DHPR is composed of a heteromultimeric complex that includes the α1s, α2, and δ, β1α, and γ1 subunits [α1s subunit  is an integral membrane protein containing four transmembrane domains, each composed of six alpha helices, acting as the pore-forming and the voltage-sensing unit. The voltage-sensing domain of α1s interacts with the γ1 subunit that regulates channel inactivation [β1a subunit is also located in proximity to the voltage-sensing domain of the α1s subunit and supports the assembly of DHPRs in tetrads, where one DHPR tetrad faces one of the four subunits of the tetrameric RyR1 channel [α1s or β1a subunits die perinatally, indicating that these subunits are essential for ECC [α1s subunit [2+ release from the SR by physically interacting with RyR1, according to “orthograde” signaling [2+ current mediated by DHPR is smaller than that measured in the presence of RyR1, suggesting that RyR1 can promote the Ca2+ channel activity of DHPR and accelerates DHPR activation. Interestingly, this activity is unique to RyR1 since RyR2 was found not to be able to restore either orthograde or retrograde signaling. Different regions in DHPR and RyR1 have been proposed to be important for their reciprocal association and regulation; the II-III loop of the α1s subunit is generally accepted to be involved in both orthograde and retrograde coupling, while the site of interaction on RyR1 has not yet been completely defined [DHPRs are L-type voltage-gated Casubunits . The α1stivation . The β1a channel ,123,124. for ECC ,126. The subunit . Despiteignaling . RyR1 isignaling ,128. In  defined ,127,129.α1s subunit of DHPR and regulates the coupling of DHPR with RyR1 [RYR1, DHPR, and STAC3 were identified in several human congenital myopathies, as summarized in STAC3 is a recently identified component of the ECC machinery. It is an adaptor protein that supports the trafficking of the ith RyR1 ,131,132.2+-binding protein (HRC) [Triadin is encoded by the TRDN gene that generates, via alternative splicing, four different isoforms named according to their molecular weight: the skeletal muscle isoforms are Trisk 95 and Trisk 51, both displaying a triadic localization; the main cardiac isoform Trisk 32  and Trisk 49, which does not localize at the j-SR in skeletal muscle fibers ,138. Triin (HRC) . Triadinin (HRC) . Triadinin (HRC) ,152. In 2+-cycling parameters in cardiac muscle [2+ release, suggesting that junctin may display a different role in cardiac and skeletal muscles [Junctin is structurally similar to triadin and, like triadin, can bind calsequestrin and the ryanodine receptor ,154,155.c muscle , but no c muscle . Similarc muscle , while i muscles .2+-binding properties. [2+ concentration [2+ concentration, front-to-front and back-to-back interactions take place. This finally results in the assembly of large ribbon-like structures, where negatively charged cavities may accommodate additional Ca2+ [2+ concentration and the presence of CASQ-binding proteins or post-translational modification [2+ depletion, it was shown that almost all CASQ is present in its depolymerized status. In this condition, progressive closure of the RyRs was reported, with a consequent reduction in Ca2+ release aimed to prevent dangerous levels of SR Ca2+ depletion. Accordingly, it has been proposed that CASQ1 depolymerization may represent the intracellular switch that induces RyR1 closing [In skeletal muscle, two isoforms of calsequestrin (CASQ) have been identified: CASQ1 and CASQ2. CASQ1 is expressed in fast- and slow-twitch skeletal muscle fibers, whereas CASQ2 is expressed in slow-twitch skeletal muscle fibers and in cardiac muscle . The twoperties. ,163. CASntration ,165. CASntration . Park annal Ca2+ . CASQ1 pfication ,170,171. closing .2+ release complex [2+ content and marked SR Ca2+ depletion during high-frequency stimulation [2+ entry that may compensate for the reduced total SR Ca2+ content [CASQ1 gene have been identified in patients affected by a rare vacuolar myopathy [In skeletal muscle, CASQs interact with RyR1, triadin, and junctin, forming a quaternary Ca complex ,173,174. complex ,180,181.mulation ,183. Recmulation ,185. Ind content ,187. Mutmyopathy  and in pmyopathy ,190.2+ and to interact with triadin [2+ levels, HRC has a pentameric structure, but when Ca2+-binding sites are saturated due to increased SR Ca2+ concentration, the protein shifts to a monomeric form [2+ homeostasis. Indeed, HRC may function as a negative regulator of RyRs and, in cardiac muscle, it was suggested to interact with SERCA pumps, acting as a negative regulator of Ca2+ re-uptake [HRC is far less abundant than CASQs, suggesting that it works as a secondary calcium-binding protein ,192. It  triadin ,194. Undric form . Althouge-uptake ,197.2+ 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 [2+ 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 [2+ ions for each hydrolyzed ATP. In vertebrates, SERCA pumps are encoded by three different genes, named ATP2A1-3. More than 10 protein variants are generated through alternative splicing occurring in the 3′-end of the main transcripts [The l-SR is mainly involved in Caent here . SERCA pembranes ,200. SERnscripts ,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 [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 [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 [2+ affinities have also been reported between SERCA2b and SERCA3 [2+ concentrations, the inhibitory effect of PLN is mitigated due to Ca2+/calmodulin kinase (CaMKII)- and/or Protein Kinase A (PKA)-dependent phosphorylation [2+, SLN uncouples ATP hydrolysis from Ca2+ transport [2+ binding protein, namely, Sarcalumenin (SAR) [2+ due to SERCA1 impairment directly contribute to muscle stiffness [ muscles ,204,205.he brain ,207,208.he brain  and cardhe brain . ATP2A3 le cells ,210. Thele cells ,212, andd SERCA3 ,214,215 d SERCA3 ,217,218.rylation ,220,221.ransport , thus prransport ,223. Thiin (SAR) , which cin (SAR) . Furtherin (SAR) ,227. Muttiffness ,229,230.2+ 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+ [+ 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+  or take up extracellular Ca2+ (a reverse mechanism) [2+ extrusion from the cytoplasm during diastole [Although SERCAs represent the key enzymes that contribute to sarcoplasmic Cafor Ca2+ . PMCAs afor Ca2+ . Among tfor Ca2+ ,234. NCXchanism) . In mammchanism) ,237, whidiastole ,239.2+ 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 [2+ 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 [2+ 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 [Entry of Caproteins . STIM1 il muscle . More reactivity ,243,244 activity ,248,249.activity ,190.2+ uniporter (MCU), a low-affinity Ca2+ transport protein , populated by several proteins and protein complexes that act as tethers between the SR and the outer mitochondrial membrane . In stri protein . It has ytoplasm . This hiniporter . From a nd FADH2 ,262, thund FADH2 . In cardmulation .2+ signaling [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 Caignaling ,266,267.2+ 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 [This review started with the statement that the SR is a specialized form of ER of muscle cells dedicated to Cabdomains . Compartbdomains ,269. Simbdomains . A less-bdomains .2+-signaling events. These junctions were identified based on the observation that nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca2+-mobilizing second messenger, induced the release of Ca2+ from a lysosome-related Ca2+ store, and this was amplified by the generation of intracellular Ca2+ waves supported by RyR3 opening, following a CICR mechanism [2+ is released with a quantal pattern; upon reaching an activation threshold, RyR3 channels open, and calcium oscillations are generated. More recently, nanojunctions between lysosomes, SR, and mitochondria have been described at the ultrastructural level in rabbit ventricular myocytes. At difference with junctions observed in pulmonary arterial myocytes, in cardiac myocytes, lysosomes are distributed with a periodicity consistent with the length of a sarcomere, allowing the association with specific areas of the SR [2+ regulation, mainly following beta-adrenergic stimulation [In eukaryotic cells, the ER is engaged in a complex network of interactions with many intracellular components, such as the nuclear envelope (NE), the plasma membrane, mitochondria, the Golgi complex, peroxisomes, endosomes, lysosomes, and lipid droplets . All theechanism . Accordif the SR . The rolmulation .2+ signaling  with functions correlated with protein synthesis and the regulation of intra-nuclear Caignaling . The NE d the NE . EM analignaling . InsP3R, by RyRs ,279, while cells . In cardexpected .Drosophila melanogaster suggests that this interaction may be mediated by a three-partner connection where amphiphysin 2 on TT binds, at the same time, Ma2/d, a protein present on both TT and SR membranes, and, at the nuclear membrane, Msp30, a protein previously described to mediate connections between the NE and the Z disks [An additional issue is represented by the SR/nuclei juxtaposition within the overall skeletal muscle architecture. In skeletal muscle, nuclei are positioned at the fiber periphery between myofibrils and the sarcolemma, in proximity to T-tubules. The positioning of nuclei is regulated by different mechanisms, including the formation of a specific microtubule network, and is critical for muscle function; indeed, improper myonuclear localization is observed in muscle diseases, such as centronuclear myopathy and muscular dystrophies . The mol Z disks ,284,285.This review provides an updated view of the structural, molecular, and functional organization of the SR in skeletal muscle, highlighting some of the specialized domains whereby this organelle supports activation of muscle contraction. The historical knowledge that the terminal cisternae of the SR engage with the T-tubules in forming the triad, which represents the first identified membrane contact site, is now expanded by evidence that in skeletal muscle, the SR participates in the assembly of at least two more types of membrane-contact sites.2+ stores under conditions of prolonged activity. The second contact site, MAM, between the SR and mitochondria, has the functional role of synchronizing the mitochondrial energy production rate with the metabolic demand of muscle fibers. Altogether, these new findings are changing the traditional view of the SR as a static organelle by depicting an unexpected dynamic ability to extend its complex structure to support muscle function.In one case, the identification of CEUs, dynamic membrane contact sites consisting of stacks of SR cisternae and the extension of T tubules, structurally and functionally different from triads, provides evidence of how skeletal muscle fibers can sustain refilling of intracellular CaAt the same time, there are several points that remain to be clarified. A major open question relates to the precise positioning of triads at the boundary of the I and A bands of the sarcomere. This highly precise pattern, evident in EM micrographs for about a half-century, is waiting for the identification of the proteins that tether triads to the sarcomere with such a regularly repetitive precision. Concerning CEU, we need to identify the molecules that support the dynamic assembly and disassembly of CEU and how Orai1 and STIM1 are recruited at these sites. Mitochondria, like many other organelles, in skeletal muscle, are localized with a well-defined pattern with respect to triads and sarcolemma, a pattern that differs in slow- and fast-twitch fibers. Additionally, in this case, the molecules that anchor mitochondria to these domains of muscle fibers remain to be identified.2+ homeostasis, but more likely, additional functions necessary to keep skeletal muscle fibers healthy and fully functional.In conclusion, we can anticipate that the above list is, certainly, still missing additional SR domains that are yet to be identified; these might probably involve activities related to the regulation of Ca