This study was performed to explore the underlying mechanisms in CaMKII-induced calpain activation to better understand heart injury. To examine the Ca2+ paradox and ischemia/reperfusion injury, isolated rat hearts were subjected to a Ca2+-free solution for 3 min, or left coronary artery occlusion for 40 min, prior to restoration of normal perfusion. Blockade of trans-sarcoplasmic reticulum Ca2+ flux using ryanodine and thapsigargin failed to prevent Ca2+ paradox-induced heart injury. In contrast, the Ca2+ paradox increased CaMKII auto-phosphorylation at Thr287, while the CaMKII inhibitor KN-62 and the Na+/Ca2+ exchanger inhibitor KB-R7943 alleviated heart injury and calpain activity. Intriguingly, the binding of μ-calpain large subunit calpain-1 (CAPN1) to phospho-CaMKII was blunted by both inhibitors. Thus, a Ca2+ leak via the ryanodine receptor is not an essential element in CaMKII-elicited calpain activation. In hearts receiving vector injection, ischemia/reperfusion caused elevated calpain activity and α-fodrin degradation, along with membrane integrity damage, similar to the effects noted in control hearts. Importantly, all these alterations were diminished with delivery of adeno-associated virus expressing mutant CaMKIIδC T287A. Ischemia/reperfusion increased CaMKII auto-phosphorylation and binding of CAPN1 to phospho-CaMKII, and facilitated the translocation of phospho-CaMKII and CAPN1 to the plasma membrane, all of which were reversed by injecting CaMKII mutant. Furthermore, the relocation capacity and the interaction of CaMKII with CAPN1 appeared to be dependent upon CaMKII autophosphorylation, as its mutant delivery increased the level of CaMKII, but did not increase membrane content of CaMKII and CAPN1, or their interactions. Together, CaMKII/calpain interaction represents a new avenue for mediating myocardial ischemia/reperfusion injury, and CaMKII likely serves as both a kinase and a carrier, thereby promoting calpain membrane translocation and activation.Previous studies indicated that Ca Ischemia/reperfusion injury induces cell death, arrhythmia, and cardiac dysfunction1. Ca2+ is essential to cardiac homeostasis. Ca2+ handling is orchestrated by an array of regulatory membrane and sarcolemmal proteins, including voltage-gated Ca2+ channel, reverse-mode Na+/Ca2+ exchange, Ca2+-ATPase on plasma membrane, the ryanodine receptor, Ca2+-ATPase on sarcoplasmic reticulum (SR), and Ca2+ uniportor on mitochondria2. Tightly regulated intracellular Ca2+ levels govern cardiac contraction in physiological conditions, whereas intracellular Ca2+ overload triggers cell death in pathophysiological states3. Multiple lines of evidence have indicated that facilitated reverse-mode Na+/Ca2+ exchange participates in myocardial ischemia/reperfusion injury5.Myocardial ischemia/reperfusion injury remains an unresolved medical problem and imposes a heavy healthcare burden2+-gated channel located on SR. Upon external Ca2+ stimulation, ryanodine receptors release Ca2+ from the SR to provoke systolic cytosolic Ca2+ peaks and maintain cardiomyocyte contractility6. Nonetheless, aberrant intracellular Ca2+ may leak through the ryanodine receptor to provoke cell death in pathological conditions such as myocardial ischemia/reperfusion injury8. Ryanodine receptor activity is tightly regulated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), a serine/threonine kinase containing catalytic, autoinhibitory, and Ca2+/calmodulin binding domains. CaMKIIδ is the predominant cardiac isoform9. Once Ca2+/calmodulin bound, CaMKII undergoes autophosphorylation at the Thr287 residue in the autoinhibitory domain and remains persistently activated even after dissociation from Ca2+/calmodulin. CaMKII is reported to be active during the early phase of postischemic reperfusion10. Previous studies have shown that CaMKII phosphorylates the ryanodine receptor at the S2814 residue in the SR, and promotes Ca2+ leak from the SR, ultimately contributing to cell death and cardiac dysfunction12. To this end, it is well known that the Na+/Ca2+ exchanger-CaMKII-ryanodine receptor cascade plays a central role in Ca2+ overload-induced injury in ischemic/reperfused hearts.The ryanodine receptor is a Ca2+-dependent protease, and the calpain superfamily possesses 15 genes in the human genome13. Several studies have revealed that over-activated calpain increases persistent Na+ current via cleaving the Na+ channel, stimulates protein kinase C, and destroys skeleton membrane proteins, α-fodrin and ankyrin-B, resulting in abnormal cell function17. μ-calpain, a heterodimer consisting of a large catalytic subunit calpain-1 (CAPN1) and a small regulatory subunit, is present in the heart. We and others have previously demonstrated that upon stress, μ-calpain becomes activated after its translocation to the sarcolemmal membrane. More importantly, inhibition of μ-calpain protects against heart injury19. Given the pivotal role of Ca2+ leak from SR in this scenario, μ-calpain activation is suspected to be a secondary effect.Ample evidence also suggests that calpain plays a vital role in ischemia/reperfusion injury. Calpain is a Ca22, and (2) calpain activity is regulated by phosphorylation modification, including protein kinase A, protein kinase C, and mitogen-activated protein kinase25. Furthermore, recent studies have demonstrated that CaMKII possesses other biological functions, such as serving as a carrier or structural component for substrate transmission27. These observations prompt us to speculate that the Na+/Ca2+ exchanger–CaMKII–calpain axis may be a primary route mediating Ca2+ overload-induced tissue injury in ischemic/reperfused hearts.Interestingly, evidence supports a more direct activation modality of μ-calpain, namely: (1) calpain destroys the ryanodine receptor during ischemia/reperfusion2+ paradox experiments. The Ca2+ paradox occurs when hearts are perfused with a Ca2+-containing solution after a period of perfusion with a Ca2+-free solution, ultimately leading to Ca2+ influx through reverse-mode Na+/Ca2+ exchange, activation of calpain, and damage to the cellular structure18. Interestingly, Ca2+ transporter proteins, ryanodine receptor and sarco/endoplasmic reticulum Ca2+ ATPase, are not involved in this event28. Therefore, we evaluated CaMKII in the Ca2+ paradox, and tested whether CaMKII-induced calpain activation is secondary to Ca2+ leak through the ryanodine receptor or functions as a more direct regulatory machinery. Subsequently, co-immunoprecipitation and immunofluorescence were performed to examine the interaction between CaMKII and calpain. Finally, the CaMKII T287A mutant was employed to discern the possible mechanism of action behind CaMKII-induced calpain activation.To this end, this study was designed to explore the underlying mechanisms in CaMKII-induced calpain activation in order to help elucidate the possible mechanism behind myocardial ischemia/reperfusion injury. To accomplish this, we conducted Ca+/K+-ATPase α1 (23565), and GAPDH (2118) were obtained from Cell Signaling Technology , Abcam  and Sigma , respectively. α-fodrin (BML-FG6090) and tetramethylrhodamine , or Alexa Fluor®488 (A32723)-conjugated secondary antibodies, were acquired from Enzo Life Sciences  and Thermo Fisher Scientific , respectively. The assay kits for LDH (KA0878), calpain (QIA120), and caspase-3 (K106-100) activity were provided by Abnova , Calbiochem  and Biovision , respectively. The Mem-PERTM Plus Kit (89842) and PierceTM BCA protein assay kit (23227) were from Thermo Scientific . All other chemicals were purchased from Sigma .KN-62 (1277), KB-R7943 (1244), ryanodine (1329), and thapsigargin (1138) were purchased from Tocris Bioscience . Antibodies against cytochrome c (4272), CaMKII (4436 and ab134041), phospho-CaMKII (12716 and ab171095), CAPN1 , Flag (F7425), cytochrome oxidase subunit IV , sodium/hydrogen exchanger 1 , Na29. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University, and all animal procedures complied with the Guide for the Care and Use of Laboratory Animals (the 8thedition) issued by the National Research Council of the United States.Male Sprague Dawley rats weighing 200–250 g were provided by the Animal Center of the Fourth Military Medical University . Animals were anesthetized via intraperitoneal administration of 3% pentobarbital sodium (60 mg/kg b. w.) before isolated heart preparation and in vivo experiments, as described previously30. The construction of recombinant adeno-associated virus (AAV) type 9 expression vector pHBAAV-CMV-MCS-3×Flag-T2A-GFP with CaMKII δC mutant T287A was performed by Hanbio Technology Co. Ltd. . The green fluorescent protein (GFP) and Flag-tagged CaMKII δC mutant were linked by a self-cleaving T2A peptide sequence, which mediates co-expression of the reporter gene GFP with the target cDNA CaMKII independently31. Viral concentration was determined to be 1.2 × 1012 viral particles per mL. The viral infection was carried out using a technique described previously29. In brief, a respiratory mask was placed on the rat’s face with 60 breaths per min and 7 mL per breath after anesthesia. The left chest and intercostal muscles between the third and fourth ribs were separated via blunt dissection. Ribs were then cut off after ligation, and the heart was exposed by pulling the ligatures out. Two sites in each side of the left descending coronary received an injection with a separation of about 2–3 mm. The injection volume for each site was 15 μL. The chest was then closed and sutured. All experiments were performed three weeks later.The cDNA of the CaMKII δC gene was synthesized based on the known cDNA sequence18. Briefly, after anesthetization and heparinization, the heart was quickly excised, and the aorta was connected to a Langendorff apparatus at a constant pressure of 80 mmHg at 37 °C. The Krebs–Henseleit (KH) perfusion solution contained the following (in mM): NaCl, 118; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 1.25; NaHCO3, 25; and glucose, 11 . The perfusates were filtered and equilibrated with a gas mixture containing 95% O2 and 5% CO2. A 20 min period was given to ensure constant mechanical properties. We preestablished that any heart with a heart rate less than 250 beats/min, or coronary flow more than 15 ml/min or less than 8 ml/min at the end of equilibration would be excluded from this study. Any heart displaying arrhythmia during this period was also discarded.Isolated heart perfusions were performed in accordance with routine laboratory methods2+ paradox studies, the hearts were, in accordance with the random number table, divided into groups as follows: (1) a control group, where the hearts received normal KH solution perfusion; (2) a Ca2+ paradox group, where the procedures were identical to our previous studies in which the hearts were perfused with a Ca2+-free solution for 3 min, followed by a normal KH solution for 30 min14; and (3) a Ca2+ paradox with drugs group, where KN-62, KB-R7943, ryanodine, and thapsigargin were introduced 1 min before and 3-min throughout the Ca2+-free solution perfusion, as well as 3 min after restoring normal KH solution. KN-62 at 3 μM and KB-R7943 at 10 μM were used to inhibit CaMKII and the Na+/Ca2+ exchanger, respectively34. Ryanodine at 10 μM and thapsigargin at 1 μM were used to block Ca2+ flux across the SR35. The sample size was estimated using statistical software PASS . Thirty rats (6 rats in each group) were used to determine myocardial injury area, and another 30 rats (6 rats in each group) were used for biochemical and histological studies. The results were interpreted by an examiner who was blind to group assignment.In CaTo determine the role of CaMKII self-phosphorylation at T287 in calpain activation in myocardial ischemia/reperfusion, rat left ventricles were randomly injected with saline (control), AAV vector, and AAV expressing the CaMKIIδC T287A mutant (CaMKII-M). Three weeks later, hearts were isolated and subjected to normal KH solution perfusion as a control (sham group). Alternatively, they received 40 min regional ischemia/reperfusion (I/R group). Briefly, a 4-0 silk suture was passed around the left coronary artery close to its origin. Forty minutes of ischemia was carried out by making a slipknot to occlude the artery and reperfusion was achieved by loosening the slipknot. In this experiment, 48 rats (8 rats in each group) were used to determine myocardial injury area, and another 48 rats (8 rats in each group) were used for biochemical and histological studies.10.Following reperfusion, the left coronary artery was religated, and 1 mL 1% Evans blue was injected to delineate the nonischemic area. Heart slices were prepared and incubated with 1% triphenyltetrazolium at 37 °C for 15 min. Myocardial injury area and risk zone were evaluated quantitatively using planimetry, as described previouslypNA), which was cleaved from the substrate DEVD-pNA. The value was read at 405 nm. Calpain activity was measured using Suc-LLVY AMC as the substrate. The released AMC was detected by fluorimetry with 360 nm excitation and 430 nm emission filters. Activity for caspase-3 and calpain was normalized in relation to the control, as described previously18.Quantification of LDH was carried out following the manufacturer’s procedures. The absorbance in the reaction mixture was read at 450 nm and the content of LDH in the coronary effluent was calculated by reference to a standard curve. Caspase-3 activity was quantified by detecting the chromophore p-nitroaniline . The excitation and emission wavelengths were 543 and 590 nm, respectively. A histologist who was unaware of the group assignment examined all sections, and the percentage of positive cells was calculated as described previously18, 5 min after reperfusion, the ischemic region of the left ventricle was excised and fixed with 10% formalin. Tissue slices with 4 μM thickness were prepared. After dewaxing, the slices were stained with antibody against CAPN1 (1:100) or phospho-CaMKII at T287 (1:100) at 4 °C overnight, followed by tetramethylrhodamine isothiocyanate or Alexa Fluor®488-conjugated secondary antibody at 1:500. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) at 2 μg/mL. Finally, a histologist, who was unaware of the group assignments examined all the slices with a confocal laser scanning microscope . Five fields for each slide were examined under a ×600 field. The degree of co-localization of CAPN1 with CaMKII is presented as a Pearson’s correlation unit using the image analysis software Image-Pro Plus. The Pearson’s correlation unit, which varies between zero and one, represents the degree of association of pixels in different channels of the confocal images. A value of zero indicates no linear relationship, whereas one reflects a perfect linear relationship between two molecules. A value above 0.7 suggests a strong positive association. To determine fluorescent protein expression, the rats were euthanized three weeks after virus injection. Frozen sections of the hearts were prepared and examined with VS-ASW-S6 .As described previously38, left ventricular tissues were homogenized with immunoprecipitating buffer containing (in mM) Tris-HCl (pH 7.4) 20, NaCl 150, EDTA 1, sodium pyrophosphate 2.5, β-glycerophosphate 1, Na3VO4 1, PMSF 1, and 1% Triton X-100. The lysates were centrifuged at 12,000× rpm for 30 min at 4 °C, and 800 μL of the supernatants were mixed with 10 μL of prewashed protein A sepharose beads in the presence of either 5 μL of rabbit anti-CAPN1 antibody for the experimental groups, or normal IgG for the control group. The mixtures were rotated constantly at 4 °C overnight. The beads were then collected by centrifugation at 3000× rpm for 2 min at 4 °C and washed three times to remove nonspecifically binding proteins. Next, the beads were suspended in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and heated at 95 °C for 5 min. Finally, SDS-PAGE was performed, and CAPN1 and T287-phosphorylated CaMKII antibodies were used for immunoblot analysis. The homogenates without bead treatment were used as the input controls.In accordance with previous studiesTM Plus Kit. To detect the content of cytosolic cytochrome c, the tissues were homogenized with buffer containing (in mM) Tris-HCl (pH 7.4) 50, sucrose 250, EDTA 1, DTT 1, and protease cocktail inhibitors. To measure other proteins, the tissues were homogenized with buffer containing (in mM) Tris-HCl (pH 7.4) 50, NaCl 150, EDTA 5, dithiothreitol 1, 1% Triton X-100, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail. The lysates were then centrifuged at 12,000× rpm for 30 min at 4 °C. The supernatants were aliquoted, and the protein concentrations were quantified with a protein assay kit. After gel electrophoresis and protein transfer from the gel to the nitrocellulose membrane, the membranes were incubated with antibodies (1:1000) of CAPN1, CaMKII, phospho-CaMKII, cytochrome c, α-fodrin, Flag, and the α1 subunit of Na+/K+-ATPase at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the proteins were probed with chemiluminescence and quantified using Quantity One software , as described previously10. Equal protein loading was confirmed by determining levels of GAPDH, COX IV or NHE1 in each group.Membrane proteins were extracted in accordance with the instructions included with the Mem-PERt tests. A two-tailed P value of less than 0.05 was considered statistically significant.All values are expressed as the mean ± standard error of the mean. Statistical comparisons were carried out using Prism 5.0 . The data exhibited normal distribution and equality of variance. A one-way ANOVA or two-way ANOVA was performed to compare the differences among several groups, as appropriate, which was followed by unpaired Student’s 2+ paradox caused severe cardiac injury as evidenced by remarkable rises in myocardial injury area, LDH release, caspase-3 activity, and the release of cytochrome c from the mitochondria to the cytosol  and thapsigargin (1 μM) failed to affect injured myocardial tissue size, LDH release, caspase-3 activity, or mitochondrial loss of cytochrome c , an inhibitor of CaMKII, attenuated Ca2+ paradox-induced cardiac injury, which manifested as reduced myocardial injury area, LDH release, and apoptosis  overtly decreased the membrane recruitment of both CAPN1 and T287-phosphorylated CaMKII Fig. . This fiTo discern if CaMKII translocation is dependent upon its phosphorylation, plasma membrane proteins were examined using a CaMKII antibody, which recognizes both non-phosphorylated and phosphorylated CaMKII. Injection of mutant CaMKIIδC T287A failed to alter membrane content of CaMKII Fig. . Moreove2+ overload-induced injury12. CaMKII phosphorylates the ryanodine receptor and provokes aberrant Ca2+ to leak from the SR, subsequently leading to hypercontracture, mitochondrial dysfunction, and sarcolemmal rupture12. We previously demonstrated that CaMKII activates calpain, another culprit factor involved in ischemia/reperfusion injury10. Thus, it is important to clarify the regulatory effect of CaMKII on calpain, which may provide insight into the mechanism of myocardial ischemia/reperfusion injury. Here, we demonstrated that Na+/Ca2+ exchanger inhibitor KB-R7943 and CaMKII inhibitor KN-62 protected hearts against Ca2+ paradox-induced injury, which was accompanied by decreased calpain activity. Our results also revealed that combined treatment with thapsigargin and ryanodine had no detectable effects. These results support the conclusion that Ca2+ influx/CaMKII is an important trigger for calpain activation and that Ca2+ leak via the ryanodine receptor is not an obligatory element in CaMKII-induced calpain activation. More importantly, our data demonstrated that: (1) calpain bound to phospho-CaMKII in both Ca2+ paradoxical and ischemic/reperfused hearts, the effect of which was negated by the CaMKII inhibitor KN-62, Na+/Ca2+ exchanger inhibitor KB-R7943, and mutant CaMKIIδC T287A; and (2) CAPN1 membrane recruitment was dependent on phospho-CaMKII in ischemic/reperfused hearts. Collectively, these results provide the first evidence that CaMKII/calpain interaction represents a new pathway mediating intracellular Ca2+ overload-induced injury. A schematic figure illustrating the CaMKII/calpain cascade in ischemia/reperfusion injury is shown in Fig. The CaMKII/ryanodine receptor cascade is considered the final instigator of intracellular Ca39. The regulation of calpain activity remains an enigma. Ca2+ is not the unique factor governing calpain activity, as Ca2+ concentrations required for its proteolytic activity in vitro are much higher than the 50–300 nM Ca2+ concentrations that exist during the diastolic period13. The results of the present investigation suggest that CAPN1 was recruited to the sarcolemmal membrane, supporting the conclusion that membrane phospholipids lower the Ca2+ concentration required for its activation41. More importantly, both mutant CaMKIIδC T287A and the CaMKII inhibitor KN-62 decreased calpain membrane localization and its activity in ischemic/reperfused hearts. These results provide evidence that CaMKII represents a new molecule that regulates calpain activity.Calpain is usually present in an inactive state in the cytosol. Our data revealed an increase of CAPN1 in the sarcolemmal membrane, in parallel with a rise of its activity and substrate α-fodrin degradation in ischemic/reperfused hearts. These results are consistent with the observation from our earlier study as well as others, supporting a culprit role for calpain in cardiac injury2+ paradox is a representative scenario that examines Ca2+ overload-induced cardiac injury. In the present study, we demonstrated that both Na+/Ca2+ exchanger inhibitor KB-R7943 and CaMKII inhibitor KN-62 were capable of reversing calpain activation to rescue the heart from the Ca2+ paradox. These results revealed that Ca2+ paradox shares similarities with ischemia/reperfusion injury, in which Ca2+ influx/CaMKII/calpain evoked heart injury10. Conversely, the Ca2+ paradox is different from ischemia/reperfusion injury in some respects. Ca2+ paradox causes a marked increase in cell necrosis, which manifests as LDH release and TTC staining. Our data showed that both thapsigargin and ryanodine failed to protect hearts against Ca2+ paradox-induced injury, which has been reported by a previous study28. Therefore, we postulate that the Ca2+ leak via the ryanodine receptor is not an obligatory element in CaMKII-elicited calpain activation.The Ca+/Ca2+ exchanger. Here, application of KB-R7943 confirmed the notion that Ca2+ influx through Na+/Ca2+ exchange serves as an important trigger for intracellular Ca2+ overload during ischemia/reperfusion5. More importantly, our data illustrated that Ca2+ influx via Na+/Ca2+ exchanger is an important determinant for CaMKII activation. CaMKIIδ is the predominant cardiac isoform9, and at least two splice variants of CaMKIIδ are detected in the heart. One is a δB isoform, which localizes to the nucleus. The other variant is a δC isoform, which has 11 amino acid deletions and exists in the cytosol9. In the present investigation, CaMKIIδC T287A reduced CaMKII activity and alleviated heart injury, compared with the AAV vector group and ischemia/reperfusion group. These results have strengthened the concept that CaMKIIδC mediates ischemia/reperfusion injury30. A previous study demonstrated that basal activated CaMKII is concentrated at the Z lines42. Our data revealed that autophosphorylated CaMKII was recruited to the sarcolemmal membrane in ischemic/reperfused myocardial cells. These results reinforce the conclusion that spatial localization determines the function of CaMKII.KB-R7943 is a widely used inhibitor of Na9. Here, the mutant CaMKII T287A reduced the interaction between phospho-CaMKII and CAPN1 but did not increase the interaction between CaMKII and CAPN1. The data did not favor a major role for CaMKII T287A mutant in competitively antagonizing the binding between endogenous CaMKII with CAPN1. In this study, we suffered from the technical difficulty of separating Flag-tagged CaMKII mutant (58 kDa) from endogenous CaMKII (55 kDa) using western blot analysis  and Ser538 in domain III (regulation domain), are modifiable by CaMKII. Therefore, the physiological significance of these residues in CAPN1 function warrants further investigation. CaMKII possesses several properties, such as mobility43, and serves as a carrier or structural component for substrate transmission27. Our data showed that self-phosphorylated CaMKII translocated to membrane upon ischemia/reperfusion challenge, and that CaMKII T287A mutation did not increase the level of CaMKII in the plasma membrane. These results suggest that CaMKII is mobility dependent upon its kinase activity. In our study, self-phosphorylated CaMKII and CAPN1 were translocated to the membrane upon ischemia/reperfusion insult, and the translocation of CAPN1 to the membrane was diminished with CaMKIIδC T287A. These observations depicted that CaMKII likely functions as a carrier, thus promoting CAPN1 membrane translocation in ischemic/reperfused hearts.CaMKII is an important kinase and auto-phosphorylation at Thr287 functions as a prerequisite for kinase activationsis Fig. . Therefo44, with a role identified for CaMKII in this process. CaMKII activates calpain, which destroys cell structure by cleaving membrane skeleton proteins, such as α-fodrin, ankyrin-B, and dystrophin10. In this work, both auto-phosphorylated CaMKII and CAPN1 were recruited to the myocardial plasma membrane. CaMKIIδC T287A blocked calpain activation and the proteolysis of α-fodrin, and therefore improved membrane integrity of myocardial cells. Thus, we conclude that CaMKIIδC/calpain causes myocardial cell injury. Previous studies have demonstrated the importance of CaMKII-dependent phosphorylation of the ryanodine receptor in myocardial ischemia/reperfusion injury by way of Ca2+ leakage11. Here, we demonstrated that CaMKII may bind with calpain. Moreover, as calpain destroys the ryanodine receptor in the SR22, our data suggest that the CaMKII/calpain may be a novel cascade for initiating ischemia/reperfusion injury. This notion is further strengthened by our data indicating that CaMKII/calpain governed Ca2+ paradox-induced injury, independent of the ryanodine receptor. It may be speculated that an interaction may exist between CaMKII/calpain and the CaMKII/ryanodine receptor cascade in ischemic/reperfused hearts, thereby engaging interaction among calpain activation, ryanodine receptor abnormality, and SR Ca2+ leak, all of which promote cell injury synergistically.Loss of membrane integrity is a salient feature in injured myocytes2+ leak via the ryanodine receptor is not an obligatory element in CaMKII-induced calpain activation and that CaMKII, likely serving as a kinase as well as a carrier, directly activates calpain to disengage membrane integrity. These findings represent a new avenue in myocardial ischemia/reperfusion injury. Further study is warranted to better clarify the mechanisms behind the regulation of calpain by CaMKII, which may provide a new strategy for heart protection.In summary, our data established that Ca


Calcium flowing through voltage-dependent calcium channels into cardiomyocytes mediates excitation–contraction coupling, controls action-potential duration and automaticity in nodal cells, and regulates gene expression. Proper surface targeting and basal and hormonal regulation of calcium channels are vital for normal cardiac physiology. In this review, we discuss the roles of voltage-gated calcium channels in the heart and the mechanisms by which these channels are regulated by physiological signaling pathways in health and disease. The influx of Ca2+ also contributes to the plateau phase of the action potential, pacemaker activity in nodal cells, and the modulation of critical cellular processes including metabolism and gene expression. Thus, a Ca2+ influx via voltage-gated Ca2+ channels in the heart links membrane depolarization to cellular functions. In this review, we will discuss mechanisms of Ca2+ handling in the heart and how dysfunctions of voltage-gated Ca2+ channels can lead to arrhythmias. Ca2+ channels are modulated by voltage, Ca2+, posttranslational modifications, and protein–protein interactions, which will also be reviewed. Finally, we will discuss existing pharmacological therapies that target voltage-gated Ca2+ channels.In the heart, calcium (Ca2+ channels exist that can be classified by membrane voltage activation (low versus high), susceptibility to pharmacologic antagonists, and rate of inactivation ; these include the T-, L-, N-, P-, Q-, and R-type channels. Of these, only long-lasting (L)- and transient (T)-type Ca2+ channels are expressed in cardiomyocytes.2 In Ca2+ channel nomenclature, the chemical symbol of calcium, Ca, is followed by a subscript “V,” denoting voltage as the primary regulator, and two numerical identifiers corresponding to the α1 subunit gene subfamily and the order of discovery within that subfamily, respectively.4 CaV1.1, CaV1.2, CaV1.3, and CaV1.4 exhibit relatively long-lasting currents and are referred to as L-type Ca2+ channels. CaV3.1, CaV3.2, and CaV3.3, which are T-type Ca2+ channels, exhibit transient Ca2+ currents and are activated at more negative potentials relative to L-type Ca2+ channels .Six classes of voltage-gated Ca2+ channels are composed of the pore-forming α1 subunit and several auxiliary subunits including β and α2δ . Four homologous domains, each with six transmembrane helices and a pore loop between S5 and S6 forming the α1 subunit. Alternating, positively charged arginine or lysine residues at every third or fourth position in S4 of each domain impart voltage sensitivity.5 The α1 subunit contains binding sites for most regulators and drugs, whereas the β, α2δ, and γ subunits contribute to trafficking, anchoring, and regulatory functions. The pore region contains binding sites for all major L-type Ca2+ channel-blocking agents including dihydropyridines, phenylalkylamines, and benzothiazepines .6Voltage-gated CaVβ1–4). The β2 subunit is the predominantly expressed isoform in the adult heart. In all of the different β subunits, the guanylate kinase (GK) and Src-homology 3 (SH3) domains are very similar, whereas the N-termini (variable region 1), the linker between SH3 and GK (variable region 2), and the C-termini (variable region 3) are quite different.9 All β subunits interact with the pore-forming α subunit via the intracellular loop between transmembrane domains I and II . In a cell-specific manner, β subunits can increase trafficking of the channel to the plasma membrane and modulate both activation and inactivation. In mice, global or cardiac-specific deletion of the Cacnb2 gene leads to abnormal heart development and embryonic death.10 Conditional deletion of the β2 subunit in adult mouse cardiomyocytes causes a 96% or so reduction in β2 protein expression, but surprisingly only a 29% reduction in Ca2+ current, with no obvious cardiac impairment,11 implying that, in adult hearts, the β2 expression may be expendable. However, interpretation of this result is ambiguous, as it is complicated by the remnant (~4%) β2 expression as well as the presence of other CaVβ isoforms expressed in adult cardiomyocytes.9 Moreover, a contrasting viewpoint was provided by a study in which short hairpin RNA–mediated knockdown of β2 in adult rat myocytes substantially diminished Ca2+ current.12There are four β-subunit genes  with multiple splice variants. Although the messenger RNAs of α2δ 1 through 314 have been identified in human myocardium, only α2δ-1 is known to bind with CaV1.2 .The α2+ channels are inactivated in both voltage- and Ca2+-dependent manners, but Ca2+-dependent inactivation is the dominant mechanism. L-type Ca2+ channels associate with calmodulin (CaM) , which modulates both Ca2+-dependent inactivation and Ca2+-dependent facilitation, where Ca2+ currents increase after repetitive stimulation.15 The importance of CaM regulation of L-type Ca2+ channels in the heart has been demonstrated by overexpressing in adult cardiac myocytes a mutated CaM protein that cannot bind Ca2+, leading to very long action potentials because of the loss of Ca2+-dependent inactivation.16 Although Ca2+ entry via the Ca2+ channel can contribute to Ca2+-dependent inactivation, remarkably, it is specifically the Ca2+ released from the sarcoplasmic reticulum via the ryanodine receptors that is the primary determinant of Ca2+-dependent inactivation.Ca2+ regulation is essential to understanding cardiac arrhythmogenesis. CaV1.2 is situated on the transverse tubules in close proximity with ryanodine receptors (RyR2), which are intracellular Ca2+ release channels located on the sarcoplasmic reticulum. Ca2+ entry through the L-type Ca2+ channels triggers ryanodine receptors to release Ca2+ from the sarcoplasmic reticulum into the cytoplasm as part of a process known as Ca2+-induced Ca2+ release .17 Ca2+ influx via Ca2+-channel current and Ca2+ release via ryanodine receptors are required for myofilament activation . Ca2+ binds to troponin C on the thin filament, allowing the myosin heads to bind to actin.18 The strength of contraction is proportional to the concentration of Ca2+ surrounding the myofilaments. In order to fully relax myocytes in preparation for the next heartbeat, the amount of Ca2+ that enters the cardiac cell during steady state must equal the amount of Ca2+ that leaves the cell.18 The reduction in cellular Ca2+ concentration is driven by Ca2+ transport via the sarcoplasmic reticulum Ca2+–adenosine triphosphate pump (SERCA) and the sarcolemmal Na+/Ca2+ exchanger . During the action potential, voltage-gated Ca2+ channels open and allow Ca2+ to flow down its electrochemical gradient, causing the plateau phase (phase 2) of the action potential . Outside of the t-tubules, L-type Ca2+ channels are also localized to caveolae—membrane invaginations important for concentrating proteins that are essential for the coordinating responses to extracellular signals—wherein Ca2+ influx can control signal transduction pathways. In ventricular myocytes, the Ca2+ current is mediated nearly entirely by the L-type Ca2+ channel, CaV1.2. In atrial and especially pacemaker cells, the L-type Ca2+ channel isoform CaV1.3, which activates at more negative potentials, contributes to the late phase 4 depolarization that underlies these cells’ automaticity .Understanding the regulation of myocyte Ca2+ current, T-type Ca2+ current has little effect on cardiomyocyte excitation–contraction coupling in the heart.19 The high density of T-type Ca2+ current in nodal cells20 and embryonic cardiomyocytes,21 however, is consistent with their putative role in pacemaker function. T-type Ca2+ channels contribute to triggered or pacemaker activity because they activate at even more negative potentials than L-type Ca2+ channels . However, they produce smaller peak Ca2+ currents and cannot substitute for L-type Ca2+ channels because T-type Ca2+ channels do not target to the sarcolemmal–sarcoplasmic reticulum junctions and therefore cannot initiate sarcoplasmic Ca2+ release.In contrast to L-type Ca2+ channel drugs, specifically dihydropyridine (prototype: nifedipine), phenylalkylamines (prototype: verapamil), and benzothiazepines (prototype: diltiazem). All three classes of drugs bind within a single overlapping region close to the pore and the proposed activation gate.23 These drugs interfere with the voltage-dependent cycling of the channel.26 The uncharged dihydropyridines, which possess higher affinity for the inactivated channel conformation (voltage- or use-dependent block), induce and stabilize inactivated channel states.27 Smooth-muscle CaV1.2 channels are more sensitive to inhibition by dihydropyridines than cardiac CaV1.2 channels because the inactivated channel states are favored in arterial smooth muscle cells due to the relatively depolarized membrane potential of these cells and the splice variant of the S6 segment of domain 1, which is specifically expressed in this tissue.29There are three main chemical classes of organic CaV1.3 is less sensitive to dihydropyridines than CaV1.2 is. Phenylalkylamines and benzothiazepines bind to the open and inactivated states with high affinity and stabilize the inactivated channel states, slowing recovery from inactivation, leading to use-dependent inhibition.31 Therefore, inhibition increases with higher heart rates, rationalizing the use of verapamil for tachyarrhythmias. Whereas verapamil and diltiazem always reduce inward Ca2+ currents, some dihydropyridines, such as (−)-BAY-K-8644 and (+)-SDS-202-791, are gating modifiers that act as agonists, increasing current amplitudes, tail currents, and single-channel open probability.27Ca2+ within the cells due to prolonged Ca2+ entry and a reduced diastolic interval for Ca2+ efflux. Moreover, some L-type Ca2+ channels become available again during the prolonged action-potential duration, and the channels reactivate, creating an inward Ca2+ influx, which can cause early afterdepolarizations.32 Increased L-type Ca2+ current also contributes to delayed afterdepolarizations and Ca2+-evoked arrhythmias, which occur after repolarization is complete and are exacerbated by sarcoplasmic reticulum Ca2+ overload. Mutations in L-type Ca2+ channels have been associated with inherited arrhythmia syndromes.Prolongation of the action-potential duration increases the loading of Ca33 is caused by the loss of voltage-dependent inactivation.33 The heterogeneous phenotype reflects the distribution of expression in the heart, brain, kidney, gastrointestinal tract, immune system, smooth muscle, testis, and pituitary and adrenal glands . The diagnosis is typically made within the first few days of life due to fetal bradycardia caused by functional 2:1 atrioventricular block. When completely expressed, Timothy syndrome is typically lethal within the first years of life. Repolarization is markedly prolonged in most patients with Timothy syndrome, with the corrected QT interval often exceeding 550 ms to 600 ms.34 Congenital cardiac defects are present in 60% of patients and cardiac hypertrophy and ventricular dilatation have been reported to occur in 50% of patients.35 Ventricular arrhythmias are the most frequent cause of death. There are no systematic studies assessing the best therapeutic strategy for patients with Timothy syndrome published to date. The available evidence supports the use of β-blockers and late Na+ channel blockers, and the use of implantable cardioverter-defibrillators for primary prevention is also reasonable.39 Close monitoring of glucose levels is essential, however, since β-blockers can mask the hypoglycemia caused by Timothy syndrome.Timothy syndrome, a multisystem disorder characterized by a prolonged QT interval and syndactyly as well as variably penetrant phenotypes of autism spectrum disorders, craniofacial abnormalities, and hypoglycemia,+ or Ca2+ currents or an increase in outward K+ currents.40 The resultant outward shift in the balance of currents active during phases 1 and 2 of the epicardial action potential allows for the already prominent transient outward K+ current to augment phase 1 repolarization. If the membrane potential is repolarized too much, L-type Ca2+ channels fail to activate, leading to a reduction in the action potential plateau predominantly in the right ventricular subepicardial cells in which the transient outward K+ current is most prominent. Loss-of-function mutations in the pore-forming α1C subunit, the β2b subunit, and the α2δ1 subunit have also been linked to Brugada, early repolarization, and short-QT syndromes.44 Agents that augment L-type Ca2+ currents, such as β-adrenergic agonists, have been shown to have therapeutic efficacy in Brugada syndrome.47Mutations in 19 genes have been identified as associated with the Brugada phenotype, causing either a decrease in inward Na48 is one of the rarest inheritable cardiac channelopathies, characterized by an accelerated cardiac repolarization. It is an autosomal-dominant disease with five identified causative genes, including three that encode for K+ channels  and two that encode for subunits of the L-type Ca2+ channels (CACNA1C and CACNB2).51 Mutations in the CaV1.2 genes CACNA1C and CACNB2b have also been associated with both idiopathic ventricular fibrillation and early repolarization syndrome.43Short-QT syndromeCACNA1D, which encodes CaV1.3, was identified in a Pakistani family with pronounced bradycardia resulting from nonconducting CaV1.3 channels.52 A loss of CaV1.3 reduces automaticity in pacemaker cells. Taken together, mutations of the core subunits of the L-type Ca2+ channels cause various cardiac syndromes and arrhythmias, including long-QT syndrome, Timothy syndrome, Brugada syndrome, short-QT syndrome, early repolarization, and bradycardia.A mutation in 18 The activity of both protein kinase A (PKA) and Ca2+-CaM-activated protein kinase (CaMKII) increases with β-adrenergic stimulation, and both kinases provoke a rise in CaV1.2 activity. The heightened activation of CaV1.2, in turn, triggers increased contractility and Ca2+-responsive signaling pathways, which contribute to the pathogenesis of heart failure and hypertrophy.54Epinephrine and norepinephrine bind to β-adrenergic receptors in cardiomyocytes, the activation of which augments inotropy, lusitropy, and chronotropy.V1.2 has remained a mystery. Experiments expressing recombinant CaV1.2 in cultured cells  have not given a clear answer, since β-adrenergic regulation is not reliably reconstituted in standard cell lines and cardiomyocytes are irrevocably altered when cultured ex vivo.56 Thus, studies are required in native systems. The failure thus far to identify any single site as essential for β-adrenergic modulation led us to propose an alternative hypothesis: that a combination of phosphorylation sites in α1C is required for β-adrenergic stimulation of CaV1.2. Since β-adrenergic regulation of cardiac CaV1.2 is conserved in vertebrates, we identified conserved PKA consensus sequences in the α1C subunit of five species: mouse, rat, rabbit, guinea pig, and human. We then generated α1C transgenic mice in which we replaced the 17 conserved consensus PKA phosphorylation sites that were not previously studied and the five conserved PKA/CaMKII phosphorylation sites that were known to be nonessential.60 Surprisingly, we found that none of these PKA consensus phosphorylation sites were necessary.61 Instead, we found that β-subunit binding to the CaV1.2 α1C subunit, but not PKA phosphorylation of β, is absolutely essential for the augmentation of Ca2+ current and cardiac contractile response to β-adrenergic−related PKA stimulation.13 These findings identify the key regulatory mechanisms impacting β-adrenergic regulation of Ca2+ influx and contractility in the heart.The molecular mechanism responsible for the β-adrenergic regulation of cardiac CaV1.2 is also a major target of CaMKII, and the resulting Ca2+-dependent facilitation of CaV1.2 current, observed as a positive “staircase” of Ca2+ current in which current amplitude increases and inactivation slows over a series of repetitive pulses, is a powerful feed-forward effect on Ca2+ signaling in the heart. It is likely that phosphorylation of both α1C and β2 subunits are required for CaMKII potentiation.63Ca2+ channels are absolutely essential regulators of intracellular Ca2+, automaticity, and contractility. The channels are regulated by a macromolecular complex consisting of core subunits and kinases, phosphatases, cytoskeletal proteins, and adaptor proteins. The dysfunction of the channels, caused by either genetic or acquired factors, is associated with heart failure, hypertrophy, or arrhythmias. Future ideal goals include providing a greater molecular understanding of the mechanisms underlying Ca2+ channel subcellular targeting, function, and modulation in cardiomyocytes in health and disease.Ca


The question of how to improve employees’ adaptive performance in dynamic environments has become a hot issue in organizational management. Although previous research has focused on the antecedents of adaptive performance, less attention has been paid to the impact of mentoring. Based on the conservation of resources theory and regulatory focus theory, this study examines the impact mechanism and boundary conditions of mentoring on protégés’ adaptive performance. In addition, through an empirical analysis of 269 samples, this study finds that mentoring has a significant positive impact on protégés’ adaptive performance. Thriving at work plays a full mediation role between mentoring and protégés’ adaptive performance, and protégés’ promotion focus moderates the relationship between mentoring and thriving at work such that the relationship is stronger among protégés with a higher promotion focus. Furthermore, the indirect relationship between mentoring and adaptive performance is stronger when protégés have a high level of promotion focus. The question of how employees can maintain high performance in uncertain and complex environments is critical for individuals and organizations. In relation to work role performance, uncertainty in an organizational context occurs when the inputs, processes, or outputs of work systems lack predictability . Models Mentoring, which is defined for the purposes of this paper as a human resource management system with both practicality and operability, is valued by the practice community . MentoriFurthermore, in terms of boundary conditions, research has been conducted on the interactions among mentoring and protégé characteristics such as learning goal orientation, self-monitoring, core self-evaluation , extroveThe research has called for non-Western samples of mentoring  to enhanSince Kram proposed the definition of mentoring in 1983, the mentoring research has gradually become an important area of organizational management. The most widely accepted definition is that mentoring is a developmental interactive relationship established between mentors and protégés in an organization in which mentors transmit knowledge, skills, and experience and provide support, guidance, and friendship to protégés .As their work role involves frequent interactions with protégés, mentors’ influences have an important impact on protégés’ career success, performance, and compensation . AccordiAdaptive performance refers to employees’ proficiency in changing behavior to adapt to job requirements and various changes in uncertain environments . From thSpecifically, the mentor provides and supplements the protégé’s work resources through guidance, thereby improving the protégé’s adaptive performance level. First, mentoring improves knowledge and skills , which iHypothesis 1. Mentoring has a significant influence on protégés’ adaptive performance.Thriving at work is a vigorous mental state that refers to the positive experience of individuals at work accompanied by vitality and learning . The twoMentoring provides protégés with a variety of work resources to help them achieve a learning state and a vigorous experience, thereby promoting the emergence of thriving at work . Some scMentoring helps the protégé experience a sense of thriving at work to achieve adaptive performance in two ways: cognitive and non-cognitive . On the Hypothesis 2. Thriving at work mediates the relationship between mentoring and protégés’ adaptive performance.Due to the potential differences in cognition and status between the mentor and protégé and because traditional Chinese culture is characterized by a high power distance, in the Chinese context, the mentor–protégé relationship is similar to a supervisor–subordinate relationship . The conAs mentioned above, research has been conducted on the interaction between mentoring and the individual characteristics of mentors or protégés  . DiffereBased on RFT, in the process of pursuing goals, the value derived from cost–benefit maximization is often less than the value derived from fit generated when the decision-making method fits an individual’s own regulatory focus. In other words, the individual will act in a way that fits his own regulatory focus; when the action direction and the regulatory focus tend to be consistent, willingness to act is enhanced and a higher level of evaluation is given to those who fit their focus . TherefoWhen the protégé holds a high level of promotion focus, he or she regulates his or her nurturance needs, which involves striving for ideals through advancement and accomplishment . He or sIn addition, the research indicates that individuals’ evaluation and use of resources depend on whether the characteristics of the resources meet individual needs. Similarly, individuals with a proactive personality or a learning goal orientation are more likely to actively seek mentoring support . The leaHypothesis 3. The protégés’ promotion focus moderates the relationship between mentoring and thriving at work such that the relationship is stronger for protégés with higher promotion focus.Based on the relationship proposed by Hypothesis 2 and Hypothesis 3, this study predicts that a protégé’s promotion focus will moderate the mediation of thriving at work between mentoring and the protégé’s adaptive performance, which constitutes a moderated mediation model.Indeed, protégés maintain a state of learning through vocational support, social psychological support, and role modeling  and reacSpecifically, when the protégé’s promotion focus level is high, the impact of mentoring on the protégé’s thriving at work will be greater, and the indirect impact of mentoring transmitted through thriving at work on the protégé’s adaptive performance will be stronger. Conversely, when the protégé’s promotion focus level is low, the relationship between the mentoring and the protégé’s thriving at work is weaker, and the positive impact of mentoring transmitted through thriving at work on the protégé’s adaptive performance will decrease.Hypothesis 4. The protégé’s promotion focus has a moderate effect on the mediation between thriving at work and adaptive performance. The indirect relationship between mentoring and adaptive performance is weaker when the protégé has a low level of promotion focus.Based on the above discussion, the conceptual model of this study is presented in The data for this study comes from 19 medium-sized enterprises in Jiangxi Province, China. Most of these enterprises are located in the ceramic manufacturing industry, the pharmaceutical industry, and the chemical industry. In these companies, formal mentoring systems have been implemented as a tool for socializing new employees. Prior to the questionnaire, we obtained the support and permission from the heads of human resources departments of the above companies. To avoid common method bias, this study uses matching data from the mentors and protégés.Mentoring function, thriving at work, and promotion focus are reported by the protégés while the adaptive performance of the protégés is reported by the mentors. On the front page of the questionnaire, we identified the purpose of this study for the respondents and guaranteed the anonymity of the survey. To make the sample more representative, 400 pairs of mentors and protégés were randomly selected for investigation in this study, and the questionnaires of 343 pairs were collected. After deleting the invalid questionnaires, 269 pairs of valid questionnaires were finally obtained (the effective recovery rate was 67.25%).Among the effective samples, 69.9% were female protégés and 83.6% were female mentors. As for the age of the protégés, the majority were 20–29 years old (approximately 85%), and 55.8% of the mentors were 30–49 years old. In terms of education, most protégés (67.3%) and mentors (54.3%) were undergraduates, accounting for 67.3%. The average tenure of the protégés and mentors was 2.57 years and 7.46 years, respectively.Except for the control variables, the items of each measure  were assessed on a five-point Likert scale ranging from 1 = strongly disagree to 5 = strongly agree.All the measurements in this study were derived from scales published in authoritative international journals. The mentoring assessment used the mentoring function questionnaire (MFQ-9) scale of 2 = 608.33, χ2/df = 2.08, CFI = 0.92, TLI = 0.91, SRMR = 0.06, RMSEA = 0.06), which indicates that the four variables have good discriminant validity, and they represent four constructs that can be used for subsequent analysis.This study uses SPSS 19.0 to test the reliability of the related scales. The internal consistency coefficient of mentoring is 0.86, that of adaptive performance is 0.90, that of promotion focus is 0.93, and that of thriving at work is 0.84. Confirmatory factor analysis was performed on four variables using Mplus7 software to determine the discriminative validity and authenticity between related variables. The fitting factors of the four-factor model are significantly better than those of other alternative models  and between mentoring and adaptive performance . A significant positive correlation is observed between thriving at work and adaptive performance , and the correlations between promotion focus and mentoring, thriving at work, and adaptive performance were r = -0.01 (p > 0.05), r = 0.20 (p < 0.01), and r = 0.18 (p < 0.01). The analysis results are consistent with the theoretical assumptions, thus laying a foundation for subsequent data analysis.p < 0.01), which confirms Hypothesis 1.To verify the research hypothesis, this study uses a hierarchical regression method to test the model. The analysis results are shown in p < 0.001). After joining thriving at work, the positive effect of mentoring on protégés’ adaptive performance was not significant  to  (Model 8), and the coefficient was still significant . According to Baron and Kenny’s test of the mediation effect, thriving at work provides full mediation between mentoring and the adaptive performance of protégés. Hypothesis 2 is thus supported.Second, the mediation effect test is considered. The positive relationship between mentoring and thriving at work in Model 2 is significant  contained 0, indicating that the mediation role is full mediation. Therefore, Hypothesis 2 is supported.∗promotion focus is significant , which indicates that promotion focus significantly moderates the relationship between mentoring and thriving at work. Hypothesis 3 is thus verified. To more intuitively show the moderating effect of promotion focus on mentoring and thriving at work, this paper draws a diagram of the moderate effect  of the School of Economics and Management in Jiangxi Science and Technology Normal University. The patients/participants provided their written informed consent to participate in this study.HZ and LZ: conceptualization and writing—review and editing. LZ: methodology. HZ: resources, writing—original draft preparation, and funding acquisition. HZ, LZ, and SR: investigation.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.


Recent studies have provided several insights into acute myeloid leukemia. Studies based on molecular biology have identified eight functional mutations involved in leukemogenesis, including driver and passenger mutations. Insight into Leukemia stem cells (LSCs) and assessment of cell surface markers have enabled characterization of LSCs from hematopoietic stem and progenitor cells. Clonal evolution has been described as having an effect similar to that of microenvironment alterations. Such biological findings have enabled the development of new targeted drugs, including drug inhibitors and monoclonal antibodies with blockage functions. Some recently approved targeted drugs have resulted in new therapeutic strategies that enhance standard intensive chemotherapy regimens as well as supportive care regimens. Besides the progress made in adoptive immunotherapy, since allogenic hematopoietic stem cell transplantation enabled the development of new T-cell transfer therapies, such as chimeric antigen receptor T-cell and transgenic TCR T-cell engineering, new promising strategies that are investigated. Moreover, genetic and molecular characterizations of AML resulted in the establishment of the 2017 ELN risk stratification  international expert panel published recommendation for the diagnosis and management of acute myeloid leukemia (AML) . This nede novo AML  whereas AML without any major cytogenetic abnormalities may emerge due to a combination of preleukemic initiating events arising in the hematopoietic stem cell (HSC) pool  pool . Strong SC) pool . LSC proSC) pool . Most caSC) pool . The traSC) pool .+ AML cases, which are defined by the presence of CD34 expression on more than 10% of blasts analyzed, contained 2 expanded cell populations with an immune phenotype of CD38—CD90—CD45RA+ or CD38+CD110+CD123+CD45RA+ both resembling normal, early and more mature hematopoietic progenitor cells (HPCs) rather than HSCs  give rise to leukemic granulocyte-macrophage progenitors (GMP-like LSC), but not vice versa, while CD38+ cells present less LSC. This suggests that the majority of CD34+ AML cells arise from HPCs that have acquired self-renewal properties, rather than from those with a direct HSC origin  mice have highlighted that 80% of CD34C origin , 20. DesC origin , a numbe38— HSPC ; CD44  and IL-1β , that drive myeloid malignancies such AML  LSCs have highlighted several cell markers, such as IL1RAP and CD25 that are overexpressed –44. Nevet models . Indeed t models , inducest models . This pat models , 49. Of t models . In addind IL-1β . Neverthnd IL-1β  whereas nd IL-1β , 52. Furnd IL-1β . Continusuch AML . IL1RAP such AML .via alteration of each cellular constituent , that overcome impaired differentiation , sinusoidal endothelial cells, osteoblasts, osteoclasts, macrophages, and immune cells, adipocytes, autonomic neurons, extracellular vesicles, extracellular matrix, and soluble factors, including cytokines and growth factors . In AML stituent –59. AML stituent , 53. Indntiation .via the fatty acid transporter CD36  proportion and lower T effector (Teff) proportion; T-cells exhaustion due to upregulation of immune checkpoint ligands and receptors; chronic inflammation and increase of inflammatory macrophage population (M1); increase of myeloid derived suppressor cell population (MDSC) and suppressive macrophage population (M2) derived from both normal progenitors and leukemia cells; and production of immunosuppressive soluble factors and metabolites , 65. Antignaling . Indeed,nd MDSCs .via the activity of immunosuppressive cells, such as Treg  fail to eliminate leukemic blasts and become senescent  as Treg . Treg le as Treg . In addi samples . Two majreported , 57. Notreported . In additivation . NK celltivation .+ T-cells and NK cells activation leading to immunogenic cell death  for 7 d and anthracycline treatment (daunorubicin 60 mg/m² per day) for 3 d , 78. Desll death . Thus chll death –82.via promoting anti-tumor response by upregulation of the antigen presentation pathway, TH1 and TH17 polarization, cytotoxic CD8 T-cells activation, Treg function reduction and immune checkpoint blockade , such as azacytidine and decitabine, are mainly used. Low dose cytarabine (LDAC) may also be used . Better blockade , 86. Conblockade . A novelblockade . Furtherblockade . Neverthgulation , 90. In gulation , 91. Of gulation , 93.via a MAC or RIC regimen without differences in the 5-year OS. However, relapses less than 6 months after primary ASCT inducing a worse OS, regardless of the treatment prescribed have been observed ; the anti-CD33 antibody drug conjugate gemtuzumab ozogamicin (GO); the IDH1/2 mutant inhibitors ivosidenib and enasidenib; the FLT3 inhibitor midostaurin in combination with chemotherapy and gilteritinib; and the BCL2 inhibitor venetoclax in combination with HMA . Daunoru+ AML. CR recovery is 13% with a median recurrence-free survival of 6.4 months. Main complications are grade 3 and 4 neutropenia (98%), thrombopenia (99%), and a few venoocclusive diseases  (+ precursors (The anti-CD33 antibody drug conjugate gemtuzumab ozogamicin (GO) was finally approved in 2017 by the FDA following a decade of controversy . CD33 o. When GOD, 0.9%) . SeveralD, 0.9%) , 119. A D, 0.9%) . A meta-D, 0.9%) . At 6 yeD, 0.9%) . CD33 execursors . Notablyecursors . The risecursors . A similecursors . Neverthecursors . Occurreecursors . Howeverecursors . Safety via calcium influx reduction, NFAT translocation and proliferation suppression  and next generation inhibitors  . Many trde novo FTL3-mutated AML and in R/R FLT3-ITD-mutated AML, has shown a CR rate of 26% and 27% respectively and a CR/CRi rate of 70% and 43% , CR, and CR/CRI (21.1% versus 10.5% and 34% versus 15.3% respectively), whereas its incidence of exposure-adjusted events was lower (19.24 versus 42.44 respectively); . Interesvia other studies , CAR T-cells or transgenic TCR Figure 2Studies pertaining to anti-CD123 unconjugated antibodies, such as talacotuzumab, have been terminated early because of issues related to toxicity . HoweverLastly, IL1RAP  blockade, using mAb81.2 and mAb3F8, which induces antibody-dependent cellular cytotoxicity and IL-1 signaling blockade in AML and CML cells provides evidence that IL1RAP may be a potent target in AML cells , 193. AlA novel adoptive T-cell transfer therapy named Chimeric Antigen Receptor T-cells (CAR T-cells), is recently available in lymphoid neoplasms, using CD19 target with promising results –197. T-cCD33 gene in HSCs prior to transplantation was suggested in order to prevent CD33 induced hematopoietic toxicity of CAR T-cells  tgTCR T-cells, which are similarly restricted to HLA-A*02:01 presentation, are reported as showing potent cytotoxicity toward not only AML cells, but also HSCs , such as RUNX1-RUNX1T1, FLT3, and NPM1, have been described as being associated with AML , resulti vaccine , 234. Ot vaccine . Moreove vaccine . However vaccine . WT1 is  vaccine  or the d vaccine . Myelope vaccine . Notablylso HSCs . In addilso HSCs , 242.via the proteomic approach. The most common HLA haplotype presenting MiHAs in European Americans seems to be HLA-A*02:01; B*44:03 (Unlike antigens presented by the HLA major histocompatibility complex (MHC), minor histocompatibility antigens (MiHAs) are encoded by germline polymorphisms. In ASCT, differences between MiHA expression in malignant and normal hematopoietic cells of patients and donors, respectively, result in GVHD but also in graft-versus-leukemia (GVL), due to HLA and Killer-cell Immunoglobulin-like Receptor (KIR) mismatches , 243. Th B*44:03 . Thus, M B*44:03 . In fact B*44:03 . Therefo B*44:03 . Lastly, B*44:03 . This st B*44:03 .As previously described, AML blasts present a deregulation of costimulatory ligand with concomitant changes in donor T-cells after ASCT. In addition, several inhibitory receptors are expressed by early differentiated memory and central memory T-cell in bone marrow of AML patients relapsing . Immune in vivo Treg depletion via immunotherapies based on bacterial toxins are also being investigated. A recent study reported preclinical results indicating that denileukin difitoxin, a diphtheria toxin, may target the IL-2 receptor and eliminate Tregs . Induction has to be followed by a consolidation therapy depending on ELN risk-stratification and age or comorbidities as follows: an intermediate dose of cytarabine (IDAC)  for younger patients in favorable- and intermediate-risk groups (18–60/65 year-old) and IDAC  for older patients in the favorable-risk group (≥60/65 year-old) . ASCT isSalvage therapy consists of IDAC with or without anthracycline described as FLAG +/- IDA . Other salvage regimens, such as mitoxantrone, etoposide and cytarabine (MEC), have shown overlapping results . The seqvia a standardized score, which is similar to that for ASCT or geriatric assessment, should be performed ; Gift 2017-03, 2018 and 2019. SFGMTC/Association Capucine Grant 2013-AO2. Supported by the MiMedi project funded by BPI France (no. DOS0060162/00) and the EU through the European Regional Development Fund of the Region BFC (no. FC0013440). French Blood Center  grants 2018 and 2019.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.


Classification of acute lymphoblastic and myeloid leukemias (ALL and AML) remains heavily based on phenotypic resemblance to normal hematopoietic precursors. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, and ignores recent advances in understanding of developmental multipotency of diverse normal hematopoietic progenitor populations that are identified by transcriptional signatures. We performed transcriptional analyses of a large series of acute myeloid and lymphoid leukemias and detected significant overlap in gene expression between cases in different diagnostic categories. Bioinformatic classification of leukemias along a continuum of hematopoietic differentiation identified leukemias at the myeloid/T-lymphoid interface, which shared gene expression programs with a series of multi or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a−CD7− subset of early thymic precursors. Within these interface acute leukemias (IALs), transcriptional resemblance to early lymphoid progenitor populations and biphenotypic leukemias was more evident in cases originally diagnosed as AML, rather than T-ALL. Further prognostic analyses revealed that expression of IAL transcriptional programs significantly correlated with poor outcome in independent AML patient cohorts. Our results suggest that traditional binary approaches to acute leukemia categorization are reductive, and that identification of IALs could allow better treatment allocation and evaluation of therapeutic options. Successful management of acute leukemia is underpinned by accurate diagnostic classification, which provides a basis for treatment allocation, risk stratification and implementation of targeted therapies . AlthougThis framework also poses difficulties for some cases of T-acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). T-ALL can be subclassified by immunogenotypic and phenotypic resemblance to either immature/ early thymic precursor (ETP), early cortical or late cortical normal T-progenitor equivalents , 7. HoweCurrent leukemia classification also takes little account of modern advances in understanding of human hematopoiesis, and the recognition of a diverse range of pluri- and multipotent progenitors, as identified by transcriptional signatures and functional assays . In partLeukemic transcriptome profiling should help to improve categorization, but traditional analytical approaches have their shortcomings. T-ALL can be reproducibly categorized according to a limited number of expression signatures that correlate with the phenotype of differentiation arrest , 29, 30.Evolutions in genomic analytical methods provide an opportunity to refine leukemia classification. We have analyzed a series of acute leukemias that comprised a high proportion of immature T-ALLs and AMLs using several complementary methods. Firstly, we show that hierarchical clustering and dimensionality reduction approaches consistently identify an AML-like subset of T-ALL. To gain deeper insight into the spectrum of differentiation arrest in these leukemias, we then used the novel Iterative Clustering and Guide Gene Selection method (ICGS). This technique, when applied to single-cell RNA-sequencing data, has been shown to infer cellular states from transcriptional data, identify modules of guide genes that are specific to these cellular developmental states in an unbiased, agnostic manner, and infer developmental relationships between these states . We shownormalize.quantiles function from the preprocessCore v1.34.0 package and batch effects between 2 independent arrays were corrected using the ComBat function (sva package). Hierarchical clustering was performed with the hclust function with distance (1-Pearson correlation) and complete clustering method. Principal Component Analysis (PCA) was performed with prcomp function. Both hierarchical clustering and PCA were performed on all probes.All computational analysis was performed in R (v.3.3.2 or above) unless otherwise specified. Data were normalized with http://www.altanalyze.org/) using HOPACH clustering, with default settings for gene expression analysis options . The gene expression filtering option was set to 2. Cell cycle genes were excluded using the most stringent parameter. From the Liu et al. pediatric cohort [ICGS was performed with AltAnalyze software v.2.1.0  for microarray and DESeq2 for RNA-Seq. Contrast matrices between selected groups are listed in Supplementary Table t-statistic for microarray data or Wald statistic for RNA-seq data. For the thymic subpopulation dataset, most variable genes across all populations were selected as the union of all the probes differentially expressed between any two populations .Differentially expressed genes were derived using the http://software.broadinstitute.org/gsea/index.jsp) using the C2.all.v6.1 collection of genesets from MSigDB (http://software.broadinstitute.org/gsea/msigdb/index.jsp) or a collection of custom genesets  were selected as genesets to be tested by GSEA. GSEA outputs were either visualized with the EnrichmentMap plugin  of Cytoscape (v.3.2.0), or with heatmaps generated with Prism software (v.7). ClueGO analysis was performed with the ClueGO plugin (v.2.1.6) of Cytoscape (v.3.2.0), using the GO Term Fusion option and otherwise default parameters.GSEA was performed with GSEA software , 9 IMD (TRD rearrangement only) and 8 IMG (TRG and TRD rearranged but absent or incomplete TRB rearrangement) leukemias. Similarly, 28/76 AML samples (40.8%) were categorized as M0-AML. Patient details are shown in Supplementary Table We performed transcriptional profiling of a series of 124 acute T-lymphoid and myeloid leukemias See . In ordeUnsupervised hierarchical clustering (HC) analysis of the expression data revealed that T-ALL and AML samples largely formed two distinct groups  or had an ETP-ALL immunophenotype (4/7 fully-phenotyped samples) , indicatWe next examined the transcriptional differences between AML-like cases and the rest of the T-ALL cohort. 2274 genes  hematopoietic progenitor transcriptional signatures that we previously reported . AML-likWe then performed transcriptional profiling of six phenotypically defined T-lymphoid progenitor groups isolated from a series of human thymuses , and comprised T-ALL cases exclusively. Conversely, Clusters 4 and 5 were effectively restricted to AML cases, with concentration of Core Binding Factor (CBF)-AMLs in cluster 5. These clusters exhibited increased expression of factors that define myeloid transcriptional modules . The intermediate Clusters 2 and 3 were characterized by heterogeneous guide gene expression, and included one third of T-ALL cases . Notably, the most immature M0 subtype AMLs were predominantly found in these two clusters , as compared with 14/48  of non-M0-AML. Also, virtually all AML-like T-ALL samples that were defined by HC  were found in either Cluster 2 (n = 4) or 3 (n = 3). ICGS therefore provides a means of classifying leukemias along a spectrum of hematopoietic ontogeny, which in our cohort included a significant number of cases at the interface between T-lymphoid and myeloid lineages. Broadly, these ‘interface’ acute leukemias (IAL) either showed no clear evidence of mature T-lymphoid or mature myeloid identity (Cluster 2), or had a partial HSPC/mature myeloid signature (Cluster 3).We then used ICGS to analyze our patient cohort. ICGS classified these leukemias into five developmental clusters that were defined by the levels of expression of a limited number of guide genes Fig.  that agap < 0.0001, all comparisons by Fisher test) and for mutations that are more frequent in immature T-ALLs [SUZ12 (p = 0.004), WT1 (p = 0.0044) and IL7R/JAK/STAT pathway members (p = 0.0364).To test whether ICGS clustering patterns correlated with mutational genotype, we performed targeted next generation sequencing (NGS) of the 79/124 cases (34 T-ALLs and 45 AMLs) where diagnostic material was available. NGS panel details are shown in Supplementary Table e T-ALLs , 40, e.gDNMT3A, IDH1 and IDH2 mutated T-ALLs being confined to cluster 2 (p = 0.0267), but PTEN mutations that normally segregate with phenotypically mature leukemias [p = 0.0287). Of note, RUNX1-mutated AMLs were exclusive to interface clusters 2 and 3 (p = 0.0015).Similarly, interface clusters were enriched for alterations in DNA methylating factors that are typical of less differentiated leukemias , 41, 42,eukemias  were sigHaving found that ICGS permits classification of acute leukemias along a spectrum of hematopoietic differentiation, we went on to more precisely characterize the transcriptional identity of individual clusters by GSEA. Analysis of the two independent T-ALL cohorts , 33 reveWithin our cohort, Cluster 1 T-ALLs were strongly enriched for mid- and late-thymic expression profiles, and negatively enriched for both early thymic and UCB HSC and myeloid progenitor signatures. AMLs in Clusters 4 and 5 had broadly converse patterns of positive and negative enrichment Fig. .Fig. 3TrTranscriptional differences in IAL Clusters 2 and 3 were less clear-cut. Cluster 2 IALs were enriched for both HSC and a series of lymphoid progenitor signatures, including MLP, LMDP, early B-cell progenitors, T-oriented CD127- Early Lymphoid Precursors (ELPs) and CD34+CD1a−CD7− early thymic cells Fig. . ClusterWe considered whether this heterogeneity might be driven by differing transcriptional contributions of T-ALLs and AMLs within each cluster. Further analysis of Cluster 2 revealed the surprising finding that while T-ALLs were mostly negatively enriched for lymphoid signatures, AMLs had expression patterns that resembled several lymphoid-competent populations, including MLPs, T-oriented CD127− and B-oriented CD127+ ELPs and early B-cell progenitors Fig. . SimilarFurther GSEA revealed that interface Cluster 2 was significantly enriched for a myeloid leukemic stem cell (LSC) transcriptional signature , and thaAs interface leukemias share expression profiles with a range of progenitors of multipotent lineage capacity, we next tested whether there was any transcriptional similarity to MPALs of either T-lymphoid/myeloid (T/M MPAL) or B-lymphoid/ myeloid (B/M MPAL) phenotype in children  and adulThe fact that interface AMLs exhibit markedly different transcription to other AML cases led us to speculate that these leukemias may have specific biology which in turn might affect clinical behavior. We therefore evaluated the outcome of interface AMLs in two independent studies , 45. To RUNX1-mutated M0-AMLs, which have previously been reported to show B-cell gene activation [In keeping with modern concepts of a hematopoietic progenitor framework that comprises a spectrum of differentiation potential, integrated transcriptional analysis of AMLs and T-ALLs revealed a continuum of leukemic developmental arrest. While AMLs and T-ALLs at either end of the spectrum were specifically enriched for the transcriptional signatures of the corresponding lineage, interface leukemias had evidence of both myeloid and lymphoid precursor gene expression, with early lymphoid signature enrichment being driven by interface AML cases. Specifically, while interface Cluster 3 AMLs had T-lymphoid transcriptional enrichment, interface Cluster 2 AMLs more closely resembled B-oriented lymphoid precursors including early B progenitors, MLPs and CD127 + ELPs , 38, andtivation . OverallTR rearrangement [WT1 and SUZ12 mutations that are typical of ETP-ALLs [PTEN alterations that are more frequent in mature T-ALLs [PHF6 mutations were always accompanied by NOTCH1 alterations in Cluster 1, 3/5 PHF6-mutated IALs (1/3 T-ALL and 2/2 AML) were NOTCH1 wild-type. This pattern was also reported in MPAL [PHF6 mutation may correlate with co-expression of other oncogenes, as shown for TLX3 [BCR-ABL leukemic background [ICGS clustering presented several important differences with accepted methods of T-ALL categorization by phenotype, immunogenotype or mutational profile , 14, 37.ETP-ALLs , 10 and e T-ALLs , 48. We  in MPAL , 49, andfor TLX3 . Interesckground . InterfaRUNX1-mutated AML-M0 cases in our cohort showed B-lymphoid identity, which is consistent with previous reports [RUNX1-mutated AMLs have recently been shown to be sensitive to glucocorticoids [RUNX1-mutated AML-M0 with AML [Our description of myeloid/T-lymphoid IALs provides support for recent proposals to define acute myeloid/T-lymphoblastic leukemia (AMTL) as a distinct diagnostic entity , but our reports . Intrigurticoids , which frticoids  and chilrticoids  might bewith AML . Finallywith AML .Supplemental DataSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S7


Recent research has shown that one of the major Ca2+ signaling pathways, store-operated Ca2+ entry (SOCE), is significantly elevated in HD. SOCE refers to Ca2+ flow into cells in response to the depletion of endoplasmic reticulum Ca2+ stores. The dysregulation of Ca2+ homeostasis is postulated to be a cause of HD progression because the SOCE pathway is indirectly and abnormally activated by mutant huntingtin (HTT) in γ-aminobutyric acid (GABA)ergic medium spiny neurons (MSNs) from the striatum in HD models before the first symptoms of the disease appear. The present review summarizes recent studies that revealed a relationship between HD pathology and elevations of SOCE in different models of HD, including YAC128 mice (a transgenic model of HD), cellular HD models, and induced pluripotent stem cell (iPSC)-based GABAergic medium spiny neurons (MSNs) that are obtained from adult HD patient fibroblasts. SOCE in MSNs was shown to be mediated by currents through at least two different channel groups, Ca2+ release-activated Ca2+ current (ICRAC) and store-operated Ca2+ current (ISOC), which are composed of stromal interaction molecule (STIM) proteins and Orai or transient receptor potential channel (TRPC) channels. Their role under physiological and pathological conditions in HD are discussed. The role of Huntingtin-associated protein 1 isoform A in elevations of SOCE in HD MSNs and potential compounds that may stabilize elevations of SOCE in HD are also summarized. Evidence is presented that shows that the dysregulation of molecular components of SOCE or pathways upstream of SOCE in HD MSN neurons is a hallmark of HD, and these changes could lead to HD pathology, making them potential therapeutic targets.Huntington's disease (HD) is a progressive neurodegenerative disorder that is characterized by motor, cognitive, and psychiatric problems. It is caused by a polyglutamine expansion in the huntingtin protein that leads to striatal degeneration via the transcriptional dysregulation of several genes, including genes that are involved in the calcium (Ca Ca2+ concentrations are strictly regulated by multiple Ca2+ channels, pumps, exchangers, and protein buffers. Under resting conditions, intracellular Ca2+ levels oscillate within the range of 100–300 nM. Upon cell activation, an increase in cytosolic Ca2+ concentrations in the range of 50–100 μM can occur in the form of microdomains by both the influx of Ca2+ ions through the plasma membrane (PM) or Ca2+ release from intracellular stores, such as the endoplasmic reticulum (ER)  is the main Ca2+ entry pathway in non-excitable cells     isoforms, STIM1, and STIM2  pump transports Ca2+ ions to the ER , which is highly selective for Ca2+, non-voltage activated, and inwardly rectifying , which is characterized by non-selective outward current with distinct biophysical features, including greater conductance than ICRAC  activity mediates ISOC  , which is around 350 kDa in size and ubiquitously expressed in the cytoplasm of all cell types  has been implicated in HD pathology, especially affecting dendritic spines. The role of SOCE and SOCE components in the formation and maturation of dendritic spines and their contribution to synaptic plasticity under physiological conditions are also discussed. Finally, recent findings are presented that support the role of molecular components of neuronal SOCE (nSOCE) and upstream pathways that regulate these processes in dendritic spine pathology in HD. Potential drug candidates are proposed that may restore normal SOCE in HD. An argument is made that nSOCE may be a novel therapeutic target for HD.The present review provides an overview of Ca2+ entry pathway in non-excitable cells is well-established. An important role for SOCE in excitable cells in the central nervous system (CNS), such as cortical pyramidal neurons  -mediated SOCE, recent studies indicated that STIM-operated TRPC1 activity mediates ISOC P2) hydrolysis upon the (S)-3,5-dihydroxyphenylglycine (DHPG)-induced activation of metabotropic glutamate receptor 1/5 (mGluR1/5) may activate TRPCs in a store-independent manner with diacylglycerol (DAG)  and ionotropic glutamate receptors, such as glutamate-sensitive N-methyl-D-aspartate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs)  and stabilizes mushroom spines that play a role in memory storage , which interacts with Ca2+ binding protein calcyclin   through RyRs , which are essential for presynaptic neurotransmitter release. In young BACHD mice that expressed full-length mHTT, an increase in striatal glutamate release was found, which was reduced to wildtype levels by Cav2.2 inhibition. Cav2.2 Ca2+ current density and PM expression also increased in these mice, which could be responsible for the elevation of glutamate release  , release of cytochrome c into the cytosol, and activation of caspases 9 and 3 and apoptosis  -2,3,4,9-tetrahydro-1H-carbazol-1-aminehydrochloride (C20H22BrClN2). This compound was able to restore disturbances in Ca2+ homeostasis and stabilize both DHPG- and CPA-induced SOCE in YAC128 MSN cultures. C20H22BrClN2 was added just 5 min before the measurements, and we concluded that it acts through the post-translational modification of proteins that are involved in SOCE, stabilization of the cytoskeleton, or the inhibition of SOCE or ER Ca2+ release channels. However, the effect of this tetrahydrocarbazole on the increase in Ca2+ release from the ER was not statistically significant, in contrast to its effect on SOCE. It specifically attenuated SOCE in YAC128 MSNs and did not affect SOCE in wildtype MSNs. We also found beneficial effects of this tetrahydrocarbazole on the function of mitochondria from YAC128 MSNs  prevents MSN spine loss in aging YAC128 co-cultures through the normalization of IP3R1 hyperactivity, suppression of abnormal ER Ca2+ release, restoration of ER Ca2+ levels, and reduction of excessive nSOCE in spines , and ruthenium red, suppressed cell death in cortical neurons that overexpressed the N-terminal fragment of mHTT, whereas the addition of the potent RyR activator 4-chloro-m-cresol (CMC) increased mHTT toxicity. In contrast, 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of IP3Rs, failed to protect these neurons  through RyRs, leading to the depletion of internal Ca2+ stores. Moreover, the expression of FK506-binding protein 12 (FKBP12), which interacts and stabilizes RyR1 by decreasing channel open permeability  stabilized the supranormal steady-state activity of IP3R1 in YAC128 MSNs in corticostriatal co-cultures  may also target mHTT in HD patients  or other disturbances in Ca2+ signaling pathways, including IP3R1, RyR, and S1R. A few known compounds may prevent dendritic spine loss in HD by stabilizing abnormal nSOCE  and S1R activity  or by attenuating supranormal IP3R1 activity . The arguments that nSOC pathways could be novel therapeutic targets for HD treatment are convincing because elevations of SOCE were observed not only in transgenic and cellular HD models but also in iPSC-based GABAergic MSNs that were obtained from adult HD patient fibroblasts. However, such proposed drug candidates or other therapeutic strategies that may act on SOCE components in HD deserve further scrutiny to provide clear evidence that they can successfully and specifically target these proteins to improve or reverse the dysregulation of Ca2+ homeostasis and signaling without causing major side effects.The dysregulation of CaMC wrote the review and discussed literature data in this paper.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


E. coli LPS and allyl isothiocyanate (AITC) induced robust Ca2+ responses in CHO-K1 cells stably transfected with mTRPA1. The amplitudes of the responses to these stimuli were significantly lower in cells treated with the cholesterol scavenger methyl β-cyclodextrin (MCD) or with the sphingolipids hydrolyzer sphingomyelinase (SMase). This effect was more prominent with higher concentrations of the raft destabilizers. Our data also indicate that reduction of cholesterol does not alter the expression of mTRPA1 in the plasma membrane in the CHO-K1 stable expression system, and that the most salient effect is that on the channel gating. Our findings further indicate that the function of mTRPA1 is regulated by the local lipid environment and suggest that targeting lipid-TRPA1 interactions may be a strategy for the treatment of pain and neurogenic inflammation.The Transient Receptor Potential ankyrin 1 cation channel (TRPA1) is expressed in nociceptive sensory neurons and epithelial cells, where it plays key roles in the detection of noxious stimuli. Recent reports showed that mouse TRPA1 (mTRPA1) localizes in lipid rafts and that its sensitivity to electrophilic and non-electrophilic agonists is reduced by cholesterol depletion from the plasma membrane. Since effects of manipulating membrane cholesterol levels on other TRP channels are known to vary across different stimuli we here tested whether the disruption of lipid rafts also affects mTRPA1 activation by cold or bacterial lipopolysaccharides (LPS). Cooling to 12 °C,  Biological membranes are dynamic and complex lipid–protein systems, forming a protective boundary around the cell and participating in multiple physiological processes . Lipid b2+-dependent crosstalk.We recently showed that mouse mTRPA1 localizes mainly in cholesterol rich domains and that disruption of lipid rafts changes the expression pattern of this protein from an extended distribution along the plasma membrane to well-defined clusters . Our datOne of the questions generated by our previous study is whether alterations in cholesterol and sphingolipid levels influence the responses of TRPA1 to other types of stimuli. This question does not have an obvious answer, because the influence of cholesterol manipulations on TRP channel function has been shown to be complex. For example, cholesterol depletion was reported not to affect the thermal sensitivity of TRPV1 , but lat2+ imaging to examine the responses of CHO-K1 cells stably expressing mTRPA1 to cold in control or after pretreatment with 1, 5 or 10 mM methyl-β-cyclodextrin (MCD). The responses were analyzed in terms of maximal amplitude of the change in intracellular Ca2+ concentration (Δ[Ca2+]). We also measured the maximal first time derivative (rising rate) of intracellular Ca2+ responses, (d[Ca2+]/dt)MAX, which is more directly related to the currents carried by the mTRPA1 channels. Control cells responded with an increase in intracellular Ca2+ concentration upon application of cooling solution MAX of the responses to cold followed a normal distribution, as the majority of cells displayed relatively small and slow responses, resulting in a mismatch between the median and the average values (2+ responses (> 500 nM) in the majority of cells . However, the correlation was low in cells treated with MCD . These observations are consistent with the weak action of these stimuli in cells treated with MCD ); 50EC is the half effective concentration and H is the Hill coefficient.The concentration-dependence curves for the stimulatory effect of AITC on mTRPA1 were fit by a Hill function of the form: