Relapsed acute myeloid leukemia (AML) is associated with the acquisition of additional somatic mutations which are thought to drive phenotypic adaptability, clonal selection and evolution of leukemic clones during treatment. We performed high throughput exome sequencing of matched presentation and relapsed samples from 6 cytogenetically normal AML (CN-AML) patients treated with standard remission induction chemotherapy in order to contribute with the investigation of the mutational landscape of CN-AML and clonal evolution during AML treatment.NPM1 p.W288fs, FLT3-ITD, NRAS p.G12D and IDH2 p.R140Q. In addition, we found two distinct clonal evolution patterns of relapse: (1) a leukemic clone at disease presentation acquires additional mutations and evolves into the relapse clone after the chemotherapy; (2) a leukemic clone at disease presentation persists at relapse without the addition of novel somatic mutations.A total of 24 and 32 somatic variants were identified in presentation and relapse samples respectively with an average of 4.0 variants per patient at presentation and 5.3 variants per patient at relapse, with SNVs being more frequent than indels at both disease stages. All patients have somatic variants in at least one gene that is frequently mutated in AML at both disease presentation and relapse, with most of these variants are classic AML and recurrent hotspot mutations including The findings of this study suggest that the relapse-initiating clones may pre-exist prior to therapy, which harbor or acquire mutations that confer selective advantage during chemotherapy, resulting in clonal expansion and eventually leading to relapse. RUNX1 . Identification of putative AML driver mutations were determined by identification of variants in established AML genes. Specifically, the Catalogue of Somatic Mutations in Cancer (COSMIC) database was interrogated to identify the most frequently recurrently mutated genes in AML.


The Ca2+ signals occurred mostly in the form of repetitive Ca2+ spikes. The probability of observing Ca2+ signals depended on the S1 concentration. The threshold was close to 70 nM, whereas at 600 nM, all cells responded. The SARS-Cov-2 nucleocapsid protein did not elicit any Ca2+ signals in any of the three cell types tested. The S1-induced Ca2+ signals in stellate cells started much faster (122 ± 37s) than those in macrophages (468 ± 68s). Furthermore, the interleukin-18 binding protein (IL-18BP) abolished the responses in macrophages without affecting the Ca2+ signals in stellate cells. The S1-elicited Ca2+ signals were completely dependent on the presence of external Ca2+ and were abolished by a selective inhibitor (CM4620) of Orai1 Ca2+ Release Activated Ca2+ channels. SARS-CoV-2 may contribute to acute pancreatitis, an often fatal inflammatory human disease. The S1-elicited Ca2+ signals we have observed in the pancreatic stellate cells and endogenous macrophages may play an important part in the development of the inflammatory process.The S protein subunit 1 (S1) of SARS-CoV-2 is known to be responsible for the binding of the virus to host cell receptors, but the initial intracellular signalling steps following receptor activation of cells in the exocrine pancreas are unknown. Using an intact live mouse pancreatic lobule preparation, we observed that S1 elicited Ca  SARS-CoV-2 is known to enter a number of cell types in the human body including the respiratory tract2, the central nervous system3 and the gastro-intestinal tract, including the exocrine pancreas4, with several reported cases of acute pancreatitis (AP)4. The ACE2 receptor is present in the pancreas4, but the mechanism by which SARS-CoV-2 can induce AP as well as the type of cells targeted by the virus are currently unknown.SARS-CoV-2 enters cells via receptor-mediated endocytosis5. However, other cell types in this tissue also play important roles, in both physiological and pathophysiological processes, including AP10, allowing preservation of the normal microscopic structure of the pancreatic environment, enables simultaneous recordings of intracellular signals i.e., cytosolic Ca2+11 in several different cell types, including stellate cells9, intrinsic nerves10 and pancreatic macrophages7.Pancreatic acinar cells dominate the exocrine pancreas and are largely responsible for the initiation of AP2+ signals in pancreatic acinar cells are well understood5, their roles in the stellate cells9 and the macrophages7 are less clear. There is, however, evidence that Ca2+ and NO signals in stellate cells amplify the pathological effects in AP7 we have studied immune cell Ca2+ signalling in response to stimulation with ATP and identified the responding cells as macrophages by immunohistochemistry with external markers specific for this cell type15, leading to inflammation and exacerbation of the patients’ conditions16, but the evidence for this has largely been limited to work on cell cultures or fixed tissue samples of the pancreas. Taking advantage of the pancreatic lobule preparation, we now report for the first time Ca2+ signals induced by the SARS-CoV-2 S1 protein in pancreatic cells.Whereas physiological and pathological CaEthical Approval: All animal studies were ethically reviewed and conducted according to the UK Animals (Scientific Procedures) Act, 1986. All experimental protocols were performed under a Project Licence granted by the UK Home Office and approved by the Animal Care and Ethics Committees at Cardiff School of Biosciences, Cardiff University. Animals were maintained in plastic cages supplied with fresh corn cob bedding, tap water, and commercial pelleted diet.7,10. The pancreas was rapidly removed, injected with standard Na+-Hepes-based solution containing collagenase and incubated for 5–6 min at 37°C. The standard solution was composed of (in mM): NaCl, 140; KCl, 4.8; Hepes, 10; MgCl2, 1; glucose, 10; CaCl2, 1 (unless stated otherwise), pH 7.3 (NaOH). All experiments were carried out with pancreatic lobules attached to the coverslip of a perfusion chamber at room temperature (22°C).Pancreatic lobules were freshly isolated from the pancreas of 5- to 7-week-old male C57BL6/J mice as previously described7. The lobules were transferred into a flow chamber and perfused with the standard solution alone or containing different chemicals as described in the experimental protocols of the result section. Cells were visualized using a Leica SP5 MPII two-photon confocal microscope, with an x63 1.2NA objective lens. The Fluo-4 excitation wavelength was 488 nm and emission was collected at 500–560 nm with resolution of 256 × 256 pixels and speed of 0.7 frames/s. Images were analysed using Leica Confocal Software . Fluorescence signals were plotted as normalized F/F0. ANOVA or Student's t-test were performed for statistical analysis.Pancreatic lobules were loaded with Fluo-4 acetoxymethyl (AM) ester at room temperature as described previously7. Mouse F4/80 Alexa Fluor 647-conjugated monoclonal rat antibodies were used to label specific surface proteins of immune cells, usually at the end of Ca2+ measurement experiments, unless otherwise stated. After blocking with 1% BSA and 10% goat serum containing PBS, the isolated pancreatic lobules were incubated for 1 h at room temperature with the selected antibody. Antibody staining was visualized by exciting Alexa Fluor 647 with 633 nm laser at 10% power and emitted light was collected at 640–700 nm. Conjugated antibody fluorescence was also overlaid with Fluo-4 staining as described in the Ca2+ measurements section. Lobules were attached to the glass coverslips covered with poly-L-lysin.Immunostaining of live pancreatic lobules was performed as previously describedSARS-CoV-2 S1 protein and Nuclear Capsid protein were purchased from GenScript or Abcam; S1 RBD (Receptor Binding Domain) was obtained from Abcam. IL-18BP was from R&D. Bradykinin (BK) was purchased from Tocris Biosciences (UK). Fluo-4 AM and Hoechst 33342 were purchased from ThermoFisher Scientific (UK). Anti-mouse F4/80 monoclonal rat antibody (CI-A3-1) [Alexa Fluor 647] were obtained from Novus Biologicals. CM4620 was provided by CalciMedica Inc., US. Other chemicals were purchased from Sigma or Calbiochem .10, pancreatic macrophages are typically sensitive to ATP or ADP and display Ca2+ signals when stimulated by these agents, whereas pancreatic stellate cells are typically insensitive to ATP, but are always responsive to bradykinin (BK), displaying Ca2+ signals when stimulated by the nona-peptide. Pancreatic acinar cells are always sensitive to acetylcholine and cholecystokinin, but never respond to stimulation with BK. They can occasionally display Ca2+ signals in response to stimulation with ATP. Nevertheless, ATP-sensitive cells are most likely to be macrophages and this can be verified after the recording of Ca2+ signals by staining with fluorescently labelled antibodies for macrophages (F4/80) . The S1-elicited Ca2+ signals in macrophages were abolished by the IL-18 binding protein IL-18BP . We have also tested the effect of the S1 receptor binding domain (RBD). As seen in 2+ signals in 69% of the macrophages (n = 16) and 71% of the stellate cells tested (n = 17).As previously reported (F4/80) –D. In the tested . The lat IL-18BP , H, wher2+ signals in any of the stellate cells or macrophages tested . In another series of control experiments, the potential effects of SARS-CoV-2 nuclear capsid protein (200 nM) was tested, but this protein never induced any Ca2+ signals in any of the cell types .We did not observe any spontaneous (in the absence of S1 or RBD) Ca2+ signals were dependent on the presence of Ca2+ in the external solution. In the absence of external Ca2+, as seen in Figures 3A and B, S1 did not elicit any change in the cytosolic Ca2+ concentration in macrophages that were responsive to ATP (n = 11) or in stellate cells (n = 9) that displayed Ca2+ signals in response to stimulation with BK. In both these cell types, it has previously been shown that an inhibitor of the opening of Orai1 Ca2+ Release Activated Ca2+ (CRAC) channels markedly reduces the duration of Ca2+ signals evoked by ATP or BK7,9. As shown in 2+ signal generation in the macrophages (n = 11) and markedly inhibited or abolished Ca2+ signals in 75% of the stellate cells tested (n = 8).We investigated whether the S1-elicited Ca2+ signals in both stellate cells and macrophages provides the first evidence for functional receptors, but not necessarily on both cell types. The much longer delay in the response to S1 in the macrophages  or TGF-ß20. Although plausible, and compatible with our data, the hypothesis that the S1-elicited Ca2+ signals in the macrophages are mediated by IL-18 from the stellate cells needs to be tested further in future experiments. The absence of Ca2+ signals in the acinar cells in response to S1 does not rule out the existence of receptors on these cells, as receptor activation could elicit effects other than Ca2+ signals but, so far, there are no data providing evidence for functional receptors on the acinar cells.There is general agreement that ACE2, the SARS-CoV-2 receptor, is expressed in the exocrine pancreas, but little is known about its distribution between the different cell typesrophages , as comprophages  showing 2+ signals, but the fact that these signals, in contrast to those evoked in the same cells by ATP or BK, are completely dependent on the presence of external Ca2+ and can be inhibited by the Orai1 CRAC channel inhibitor CM4620, suggests a mechanism of generation that is somewhat different from that of the physiological agonists.At this stage there is no information about the intracellular steps leading to the generation of the Ca2+ and NO signalling in pancreatic stellate cells9,10 and Ca2+ signalling in pancreatic macrophages7, we have proposed that AP, although initiated in the acinar cells, progresses to a severe and often fatal disease state through necrotic amplification loops, sustained by excessive Ca2+ entry through CRAC channels in stellate cells, macrophages and acinar cells. We have specifically provided evidence for a number of interaction pathways between acinar cells and stellate cells and between acinar cells and macrophages5. The new data presented here suggest a further pathway, not hitherto recognized, between stellate cells and macrophages in which IL-18–secreted by stellate cells in response to activation of SARS-CoV-2 receptors—acts on macrophages to generate Ca2+ signals in these cells. IL-18 levels are elevated in AP, and it has previously been suggested that this agent may play a role in acute and chronic pancreatitis together with several other interleukinsBased on our previous studies of Ca2+ signals in macrophages and stellate cells, demonstrated in this study, would be likely to exacerbate an otherwise mild case of AP initiated, for example, by the primary action of bile acids or a combination of ethanol and fatty acids on the acinar cells, and could help to explain the known cases of severe AP associated with SARS-CoV-2 infection4,24. Our finding that the S1- and RBD-elicited Ca2+ signals can be inhibited by an Orai1 CRAC channel blocker, is a further argument in favour of this pharmacological treatment against AP5,25.The S1 and RBD-elicited Ca


The following characteristics of mitochondria are involved in the physiological and pathological regulation of cells: mitochondria mediate calcium uptake through calcium uniporters; mitochondria are regulated by mitochondrial dynamic related proteins  and form mitochondrial networks through continuous fission and fusion; mitochondria supply NADH to the electron transport chain through the Krebs cycle to produce ATP; under stress, mitochondria will produce excessive reactive oxygen species to regulate mitochondria‐endoplasmic reticulum interactions and the related signalling pathways. Both SOCE and mitochondria play critical roles in mediating cardiac hypertrophy, diabetic cardiomyopathy, and cardiac ischaemia‐reperfusion injury. All the mitochondrial characteristics mentioned above are determinants of SOCE activity, and vice versa. Ca2+ signalling dictates the reciprocal regulation between mitochondria and SOCE under the specific pathological conditions of cardiomyocytes. The coupling of mitochondria and SOCE is essential for various pathophysiological processes in the heart. Herein, we review the research focussing on the reciprocal regulation between mitochondria and SOCE and provide potential interplay patterns in cardiac diseases.Store‐operated Ca Voltage‐gated Ca2+ channels (VGCCs) are the most studied plasma membrane (PM)‐anchored channels, and store‐operated Ca2+ entry (SOCE) channels on PM have received increasing attention.2+ flows into electrically excitable and non‐excitable cells via VGCC and SOCE, respectively. Cardiomyocytes are excitable cells, and previous studies have mainly focussed on their VGCCs. Recently, SOCE has received increased attention in cardiomyocytes; SOCE‐dependent Ca2+ signalling is crucial for the development of the heart and the pathological processes of cardiovascular diseases.Since cells use Ca2+ from ER stores.2+ concentration in the cytoplasm mediates the contraction and relaxation of cardiomyocytes. The intracellular calcium concentration is precisely regulated to avoid cellular injury. The calcium balance is maintained by Ca2+‐permeable channels (mainly DHPR and RyR2), calcium pumps, and Na+‐Ca2+ exchangers (regulating intracellular Ca2+ concentration via exchange of Ca2+ and Na+).2+ level during the activation of voltage‐dependent calcium entry. Calcium oscillations during excitatory contractile coupling of cardiomyocytes do not change the overall intracellular calcium concentration. Compared with DHPR/RyR2, SOCE mediates sustained intracellular calcium influx resulting in a moderate increase in intracellular calcium concentration.L‐type VGCC, a plasma membrane‐localised voltage‐dependent calcium channel, is critical for the excitation‐contraction coupling of the heart.2+ release, causing persistent SOCE events and continuous cytoplasmic Ca2+ entry. IP3 is activated in the heart under pathological conditions. G protein‐coupled receptors, the plasma membrane receptors, are activated when binding to endothelin I and angiotensin II , leading to phospholipase C‐dependent release of IP3 from phosphatidylinositol 4,5‐bisphosphate; then, IP3 binds to the IP3 receptor (IP3R), which is an ER Ca2+‐release channel, to activate it2+ depletion from the ER lumen to the cytosolInositol 1,4,5‐triphosphate (IP3) activation is critical for ER Ca2+ storage organelles; this function is enabled by the mitochondrial Ca2+ uniporter (MCU).In addition to their central role in metabolism, mitochondria are CaAccumulating evidences supports the existence of reciprocal regulation between mitochondria and SOCE in the physiological and pathological processes of the heart.22+ entry from the extracellular space into the cytosol.2+ release‐activated Ca2+ channel (CRAC) is a Ca2+ selective channel formed by Orai1SOCE enables Ca2+ influx from extracellular space to cytosol. Orai1, Orai2, and Orai3 are subfamily members of Orai. Orai proteins form Ca2+‐selective (PCa/PNa >1000) channels.2+, Na+, Sr2+, and Ca2+. TRPCs contain six subgroups: TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7.Orai and TRPCs are PM‐anchored proteins; the channels formed by these two proteins induce Ca2+ from the ER.2+ concentration in the ER; the answer to this question was obscure until the discovery of STIM1, an endoplasmic Ca2+ depletion sensor protein. STIM1 oligomerization is initiated upon the release of Ca2+ from the ER lumen. STIM1 and STIM2 are two subfamily members of the STIM. STIM1 has been well studied and has been proven to be a Ca2+ sensor.SOCE events are activated by the discharge of Ca2+ in the ER), Ca2+ inhibits STIM1 when binding to its EF‐hand domain.2+ content in the ER can promote STIM1 oligomer formation and expose the activating region within the coiled‐coil domain.2+ influx2+ levels in the ER lumen, STIM1 oligomerizes and interacts with SOCE channels to elicit Ca2+ influx. Ca2+ entry will persist as long as Ca2+ levels remain low in the ER.STIM1 is a transmembrane protein of the ER; it consists of an intra‐ER EF‐hand domain, a transmembrane domain, a sterile α‐motif domain, an extra‐ER distal lysine‐rich cytosolic tail, and an extra‐ER coiled‐coil domain. In the resting state  uptakes Ca2+ from the cytosol to the ER lumen. Following SOCE activation, SERCA pumps are aggregated around STIM1 oligomers; SERCA re‐uptake Ca2+ ions as soon as they cross the SOCE channels and reach the cytosol, avoiding the diffusion of Ca2+ ions in the cytosol and efficiently refilling the ER Ca2+ store  and plasma membrane Ca2+ ATPase (PMCA) are capable of inducing Ca2+ extrusion from the cytosol.2+ affinity but a high capacity for Ca2+ transport, whereas PMCA exhibits the opposite properties.2+ variations (both amplitude and content changes) have been observed when cardiomyocytes undergo excitation‐contraction coupling, mainly achieved by dynamic regulation of NCX, but not by PMCA.2+ elevation elicited by SOCE in the cytosol. Therefore, PMCA activation is the main factor that mediates Ca2+ extrusion during SOCE.Both the Na32+ influx from regions adjacent to Ca2+ entry sites with high Ca2+ concentrations, referred to as calcium concentration microdomains (CCMs).2+ level in CCMs is high, it inhibits the activity of SOCE channels via a process called Ca2+‐dependent SOCE inactivation. This property of the SOCE channel is crucial for the interplay between SOCE and mitochondria.SOCE induces Ca2+, relieving the inhibitory effect of incoming Ca2+ on SOCE activity.2+ signalling and ROS production in cells.2+ signals and ROS are mainly handled by the mitochondria and are linked to SOCE activity.2+ ([Ca2+]m) handling and ROS generation. Moreover, location, motility, and dynamics of mitochondria are also determinants of SOCE activity.Mitochondrial function is closely related to the activity of SOCE.2+ influx can affect the concentration of [Ca2+]m and subsequently modulate mitochondrial energy metabolism (ATP production) and mitochondrial stress response (ROS generation). MCU responds to increased SOCE activity and initiates [Ca2+]m uptake to accumulate Ca2+ within the mitochondrial matrix2+]m concentration stimulates oxygen consumption to accelerate ATP production and ROS generation.Conversely, SOCE plays a vital role in regulating mitochondria.3.12+ channels (DHPR) in the T‐tubule membrane come very close to SR localised Ca2+ release channels (RyR2) to form a calcium release unit (CRU)  and PM‐anchored proteins (Orai1), together with the ER and PM.2+]m uptake and, in turn, sub‐PM mitochondria are capable of modulating SOCE activity.Conventionally, it was believed that mitochondria, localised in the vicinity of CCMs, directly regulated SOCE activity. Following the SOCE event, mitochondria were thought to take up Ca2+ diffusion between individual mitochondria via inter‐mitochondrial junctions, it is still not clear whether the whole mitochondrial network can freely share the Ca2+ ion. Presumably, as with the segregation of mitochondrial damage, SOCE‐related mitochondrial Ca2+ uptake may also be limited without spreading throughout the mitochondrial network  and interfibrillar mitochondria (far from SOCE channels) in cardiomyocytes.3.23.2.12+, calcium depends on transporters to accumulate in the mitochondria. MCU, a Ca2+ transporter, can be activated by increased [Ca2+]c (cytosolic Ca2+) and subsequently uptake Ca2+ from the cytoplasm to the mitochondrial matrix; uncoupling protein 2 (UCP2) is also a Ca2+ transporter on the mitochondrial inner membrane. Their specific roles in Ca2+ handling differ. MCUs can respond well to increased Ca2+ levels regardless of the source of Ca2+, whereas UCP2 uptake from the intracellular released Ca2+ but not that of incoming through SOCE channels.2+ transporters (MCU and UCP2) are essential for maintaining SOCE activity.Since the mitochondrial inner membrane is impermeable to Ca3.2.22+ uptake transporters (MCU and UPC2), Ca2+ extrude transporters, and Ca2+‐H+‐ exchanger (Letm1) are essential for maintaining [Ca2+]m homeostasis. SOCE can mediate the upregulation of MCU, thereby controlling mitochondrial Ca2+ uptake and regulating mitochondrial metabolism.+‐Ca2+ exchanger, and Ca2+‐H+ exchanger respond to the SOCE event.It is known that Ca2+]m uptake.2+]m uptake is essential for mitochondrial ROS (mtROS) generation. A study has shown that MCU is associated with mtROS production, thereby promoting cellular skeletal response during wound healing in Caenorhabditis Elegans.SOCE promotes the activation of MCU that facilitates [Ca2+]m uptake regulates ATP production via oxidative phosphorylation. In mitochondria, calcium ions activate three key enzymes related to the TCA cycle: pyruvate dehydrogenase, ketoglutarate dehydrogenases, and isocitrate dehydrogenases.2+ concentration in the mitochondrial matrix to increase NADH availability, drive electrons to flow through the respiratory chain, and ultimately enhance ATP synthesis. In addition, the SOCE‐mediated increase in cytosolic Ca2+ ([Ca2+]c) enhances the activity of aralar1 and citrin  to accelerate the supply of α‐ketoglutaric acid (α‐KG) to the tricarboxylic acid cycle (TCA cycle), and ultimately promote ATP synthesis.MCU‐dependent  c. It is well established that increased [Ca2+]c levels activate calcineurin/Drp1 and CaMKIα/Drp1 pathways to promote mitochondrial fission.Conversely, SOCE activity has a profound impact on mitochondrial dynamics. STIM1 knockout promotes mitochondrial fusion and increases metabolic activity in mouse embryonic fibroblasts,44.12+ in neonatal cardiomyocytes. We infer that Orai1 but not TRPCs mediated SOCE events that contributed to the postnatal development of cardiomyocytes.SOCE is essential for both physiological and pathological heart regulation. The protein expression of the STIM, Orai, and TRPC subfamilies is influenced by the corresponding cell types. Determining the characteristics of the expression of these proteins in cardiac myocytes is necessary to understand the specific mechanism of myocardial SOCE. STIM1 and STIM1L (a splice variant of STIM1) transcripts are both present in neonatal cardiomyocytes but diminish with maturation. STIM1 and STIM1L have been found to be upregulated in myocardial cells under stress (e.g. phenylephrine treatment),4.22+ is essential for the excitation and conduction of the heart. It was confirmed that STIM1 was expressed in coronary sinus cardiomyocytes (CSCs) in a tract from the sinoatrial node to the coronary sinus.2+ that effectively elicits the SOCE event in mouse ventricular myocytes.2+ waves. The arrhythmogenic role of SOCE was verified by another research group using a genetic murine model of arrhythmic disease . They demonstrated that transient Ca2+ entry events comprise cardiac SOCE and can be effectively prevented by SOCE inhibition.The regulation of Ca5TRPCs and Orai1 are upregulated, and STIM1 is activated, during the progression of cardiac hypertrophy.Hypertrophic transformation of the heart can be divided into three stages: developing hypertrophy (stage 1), compensatory hypertrophy (stage 2), and overt heart failure stage 3)..3). This+]m levels to induce mtROS‐NF‐kB signalling and, in turn, mtROS‐NF‐kB signal enhances SOCE activity to form a positive feedback loopDuring the progression of cardiac hypertrophy, mtROS generation is moderately increased that subsequently activates its downstream signalling pathway, NF‐kB.In hypertrophic cardiomyocytes, mitochondrial metabolic status (which activates AMPK) and mitochondrial ROS generation (which activates NF‐κB) oppositely modulate SOCE activity. AMPK attenuates and NF‐κB facilitates SOCE activity. In short, mitochondria can either promote or inhibit SOCE activity in the hypertrophic heart.66.1+/− mice develop dilated cardiomyopathy that cannot compensate for the overload and results in an earlier death.R93W knock‐in mice that express a non‐functional Orai1 channel protein exhibited markedly swollen mitochondria with an abnormal cristae structure in the heart.Changes in the components of the SOCE machinery were observed in patients who were diagnosed with heart failure in the NYHA III‐IV class. Orai1 was decreased by 30% in failing myocardium, whilst Orai2 and Orai3 expression remained normal. The STIM2.1 levels are significantly reduced to enhance SOCE activity in end‐stage heart failure.2+ uptake, and SOCE‐dependent activation of Ca2+‐regulated transcription factor promotes MCU expression, in turn controlling the Ca2+ uptake capability of mitochondria and hence regulating mitochondrial metabolism.In the dilated heart, the metabolic substrate preference of mitochondria converts fatty acids to glucose. Interestingly, SOCE events dramatically decrease during the transition of the heart from compensatory hypertrophy to dilated heart failure. Thorough investigations of the relationship between SOCE and mitochondrial metabolism have been conducted to elucidate their reciprocal regulation in heart failure. In the failing heart, compromised SOCE activity reduces lipid metabolism in a mitochondria‐dependent manner that is crucial for the mobilisation of fatty acids from lipid droplets and mitochondrial fatty acid oxidation; SOCE controls cAMP‐dependent induction of PGC‐1α/PPARα expression and lipolysis.6.22+‐dependent myofilament activation, also referred to as excitation‐contraction coupling. NCX‐ and SERCA‐dependent dissipation of CCMs is a prerequisite for cardiac excitation‐contraction uncoupling. Therefore, the malfunction of the dissipation‐machinery of CCMs causes the retention of Ca2+ in the cytoplasm, resulting in HFpEF. Recent studies have demonstrated that SOCE and mitochondria contribute to HFpEF; SOCE improves the elevation of cytosolic Ca2+ and the development of cellular hypercontracture2+]i that may inhibit the diastolic function of cardiomyocytes.2+ entry during the diastolic period of cardiomyocytes, resulting in sustained elevation of cytosolic Ca2+ and HFrEF.CRU, formed by DHPR and RyR2, elicits the accumulation of CCMs adjacent to myofilaments, providing a coupling mechanism that enables Ca7Diabetes is characterised by hyperglycaemia and insulin resistance. The attachment of O‐linked N‐acetylglucosamine to proteins (O‐GlcNAcylation) is observed under hyperglycaemic conditions. In cardiomyocytes, hyperglycaemia‐mediated STIM1 O‐GlcNAcylation decreases SOCE activity2+]m uptake is vital for maintaining SOCE activity, and MCU is critical for inducing [Ca2+]m uptake.2+]m uptake.2+ counteracting the inhibitory effects of Ca2+ on SOCE activity, the paucity of ATP attenuates the SOCE activity in the diabetic heart.m accumulation causes overproduction of mtROS and activation of apoptosis pathways.2+]m‐mtROS pathway to deteriorate IRI. Intriguingly, I/R‐mediated mtROS generation can increase the sensitivity of IP3R to maintain empty ER Ca2+ stores, thus stabilising the opening of SOCE channels.During I/R, SOCE is activated to induce Ca2+ overload and promote mitochondrial fission in response to I/R. The increased [Ca2+]c during I/R activates calcineurin that targets drp1 (S637) for its de‐phosphorylation.2+]c, phosphorylates Drp1; both calcineurin and CaMKIα promote translocation of Drp1 to mitochondria to induce mitochondrial fission.2+]c‐Drp1 signal to promote mitochondrial fission ; Supervision (lead). Jinliang Nan: Conceptualization (lead); Writing‐original draft . Jiamin Li: Writing‐original draft . Yinuo Lin: Writing‐review & editing . Muhammad Saif Ur Rahman: Writing‐review & editing . Zhengzheng Li: Writing‐review & editing .


Musculoskeletal disorders (MSD) are a common health problem among dentists. Dental treatment is mainly performed in a sitting position. The aim of the study was to quantify the effect of different ergonomic chairs on the sitting position. In addition, it was tested if the sitting position of experienced workers is different from a non-dental group.A total of 59 (28 m/31f) subjects, divided into two dentist groups according to their work experience (students and dentists (9 m/11f) < 10 years, dentists (9 m/10f) ≥ 10 years) and a control group (10 m/10f) were measured. A three-dimensional back scanner captured the bare back of all subjects sitting on six dentist’s chairs of different design. Initially, inter-group comparisons per chair, firstly in the habitual and secondly in the working postures, were carried out. Furthermore, inter-chair comparison was conducted for the habitual as well as for the working postures of all subjects and for each group. Finally, a comparison between the habitual sitting posture and the working posture for each respective chair (intra-chair comparison) was conducted . In addition, a subjective assessment of each chair was made.For the statistical analysis, non-parametric tests were conducted and the level of significance was set at 5%.When comparing the three subject groups, all chairs caused a more pronounced spinal kyphosis in experienced dentists. In both conditions , a symmetrical sitting position was assumed on each chair.The inter-chair comparisons showed no differences regarding the ergonomic design of the chairs. The significances found in the inter-chair comparisons were all within the measurementerror and could, therefore, be classified as clinically irrelevant.The intra-chair comparison  illustrated position-related changes in the sagittal, but not in the transverse, plane. These changes were only position-related (forward leaned working posture) and were not influenced by the ergonomic sitting design of the respective chair. There are no differences between the groups in the subjective assessment of each chair.Regardless of the group or the dental experience, the ergonomic design of the dentist’s chair had only a marginal influence on the upper body posture in both the habitual and working sitting postures. Consequently, the focus of the dentist’s chair, in order to minimize MSD, should concentrate on adopting a symmetrical sitting posture rather than on its ergonomic design.The online version contains supplementary material available at 10.1186/s12891-021-04334-1. It has been shown that dentists who work in a sitting position are more likely to report severe lower back pain than those who alternate between sitting and standing , 2. SincFurther factors such as an unfavorable posture , 23, 24,This can lead to muscular fatigue and strain on the peripheral nervous system due to recurring movements and static positions held for long periods of time. Over time, these can also lead to pathological changes in the musculoskeletal system and the spine , 29, 30.As a result of these work-related complaints the field of dental ergonomics has generally received increasing attention in recent years. In the field of dental ergonomics, efforts are being made to reduce these prevailing physical strains by, among other things, attempting to modify the dentist’s chair layout. According to the manufacturer, the dentist’s chair should allow the user to sit in a stable, relaxed position with a straight back, without twisting the spine or turning the head and with a good view of the working area , 36. ErgRelated studies such as those by Gandavadi et al.  and DablM. deltoideus, M. biceps brachii, M. triceps brachii, M. sternocleidomastoideus, M. trapezius  and M. rectus abdominis) while sitting on six different chairs in five different posture positions. Although Haddad , 4746, 4Videorasterstereography has already been confirmed as a suitable method for recording upper body posture –44 and, Six different chairs were selected for this study. They differed in their design and underlying ergonomics; these are summarized in Table Chair 1,2,3, 5 and 6 were chosen since they are the models commonly used in German dental practices and universities. Only chair 4, the Swopper, is not designed for the field of dentistry. It is an ergonomically designed chair whose construction is flexible enough to allow “moving sitting” (bouncing). Body muscles can thus be moved unconsciously, actively reducing a static sitting posture.The investigation was conducted at the Institute of Occupational Medicine, Social Medicine and Environmental Medicine at Goethe-University Frankfurt/Main (Germany). One measurement took about 2 s. Since we performed a high number of measurements on the 6 chairs, the measurement of each subject took approx. 1 hour.Based on the questionnaire used in the study on musicians’ chairs , the subPrior to the initial study, the questionnaire was evaluated in 10 dental students.In this context, the following questions, among others, should be answered in relation to each chair: How did you like the chair designs you just tried? What did you particularly like about the best chair? Here, the three main criteria mentioned by the test persons were the comfort, the clearly defined sitting position of some chairs and the ergonomic design.Prior to the body posture measurements being taken, the backrests of chairs 3, 5 and 6  were removed for the duration of the measurements in order to enable the measurement of the back down to the landmark rima ani. Dable et al.  confirmeThe order of the chairs and the sitting postures were randomized. Each subject was measured in two different sitting postures  on all six chairs Fig. . For theThree consecutive measurements were performed from which the mean value was calculated.Additionally a subjective assessment of chair comfort was conducted which based on the school grading system, with 1 representing the best and 6 the worst rating.The program “BiAS 11.08”  was used for the statistical analysis. Firstly, normality assumptions were tested by the Kolmogoroff-Smirnoff-Lilliefors test. Since the data did not show a normal distribution, non-parametric tests were used.Secondly, in order to compare all chairs for each back parameter, either in the habitual sitting position or in the dental working position, the Friedman-test was carried out with subsequent multiple pair comparisons.All three groups were compared with the Kruskal-Wallis test followed by multiple pair comparisons. To compare both postures, the Wilcoxon-matched-pairs-test was used.p-values were corrected by Bonferroni-Holm correction. The subjective assassement of chair comfort was calculated by the Chi2 test for the contingency table.All The significance level was 5%.Table p ≤ 0.02). The thoracic bending angle was significant (p ≤ 0.02–0.04) for chairs 1, 5 and 6, while the lumbar bending angle was significant in chairs 2, 3, 4 and 5 (p ≤ 0.001–0.03). The kyphosis angle was significant for chairs 3, 4 and 5 (p ≤ 0.001–0.04), whereas the lordosis angle was significant in chairs 2, 3 and 4 (p ≤ 0.001–0.05). The frontal trunk decline was only significant in chair 1 and the maximum lateral deviation was significant only for chair 3 (p ≤ 0.01–0.05).The parameter trunk length S was significant for all six chairs (p ≤ 0.01–0.02) in the dental working posture. The parameters of the thoracic bending angle and kyphosis angle showed significances in all chairs, except in chair 3 (p ≤ 0.01–0.05). Furthermore, for each parameter only one significance was found: trunk length D in chair 1, sagittal trunk decline in chair 4, standard deviation rotation in chair 5, lordosis angle in chair 4, scapular height in chair 4, scapular angle left and right in chair 3 and pelvis rotation in chair 5 (p ≤ 0.001–0.05).As in the habitual sitting posture, the parameter of trunk length S was also significant for all chairs . In group 1, the trunk length D, trunk length S, sagittal trunk decline and axis decline (p ≤ 0.001–0.02) were significant. Significances were found in group 2 for the trunk length D, trunk length S and sagittal trunk decline (p ≤ 0.001). Within group 3, differences were found in the trunk length D, trunk length S, sagittal trunk decline, lumbar bending angle, kyphosis angle, pelvis height in degrees and pelvis height in mm (p ≤ 0.001–0.05).The comparison of the chairs in all groups: trunk length D, trunk length S, sagittal trunk decline, axis decline, scapular distance, scapular angle left, pelvis distance, pelvis height in degrees and pelvis height in mm (p ≤ 0.001–0.05). In Group 1, only the pelvis distance was significant (p ≤ 0.04), while group 2 had significances for the parameters of trunk length D, trunk length S and sagittal trunk decline, as well as for pelvis distance, pelvis height in degrees and pelvis height in mm (p ≤ 0.001–0.05). Group 3 showed significant deviations in trunk length D, trunk length S, standard deviation rotation and pelvis distance (p ≤ 0.03).The following significant differences between the chairs could be observed in habitual sitting posture and the working posture within each chair. This was calculated, on the one hand, for all test persons and, on the other hand, for each group. All significances that occurred had a value of p ≤ 0.001.Table posture comparison in all groups showed statistically significant p-values between the habitual sitting and working postures on all six chairs for trunk length D, trunk length S, sagittal trunk decline, thoracic bending angle, scapular distance and scapular angle right.The For chairs 2, 4, 5 and 6, the parameter of standard deviation rotation was significant. The kyphosis angle showed significances for chairs 2, 3, 4 and 5, while the lordosis angle was different in chairs 2 and 4. For the parameter of scapular height, only chair 1 showed a significant difference. In the scapular angle left, significances were found for chairs 3 and 4. Finally, for the parameter of pelvis distance, significance was found for chairs 2 and 5.group 1 there were deviations in the parameters of sagittal trunk decline, thoracic bending angle and scapular distance for all 6 chairs. Only chairs 2, 3 and 4 differed in the parameter of trunk length D. The parameter of kyphosis angle was significant for chairs 3, 4 and 5, while trunk length S was significant for chair 2 and the lordosis angle for chair 3.Within group 2, all six chairs showed significant values for the parameters of sagittal trunk decline, thoracic bending angle and scapular distance. The parameters of trunk length D and trunk length S were significant for all chairs except for chair 4, whereas only in chair 2 did the scapular angle left show no significance. Finally, standard deviation rotation had a significance only in chair 6.In group 3, the parameters of sagittal trunk decline, thoracic bending angle and scapular distance showed significant differences between the habitual sitting and working postures within all chairs used. Standard deviation rotation had significance in chairs 5 and 6. Some parameters had only one significance each: trunk length S for chair 2, kyphosis angle for chair 5, scapular angle left for chair 3, pelvis distance for chair 2 and pelvis rotation for chair 5.In 2 test. The corresponding distribution of scores is shown in the form of a contingency table in Table With regard to the group differences of the subjective assessment per chair, no significant group differences are shown in the ChiThe analysis showed that the ergonomic chair layout did not have a clinically relevant effect on the upper body posture, either in the habitual sitting or working postures. There was also no clinically relevant correlation with regard to the choice of profession or the work experience of the dentists. This is confirmed by the subjective assessment of each chair in the group comparison. The sitting position alone, habitual or the anteriorly inclined, dentally-idealized treatment position, was found to be the decisive factor.Only position-related changes in the sagittal, not in the transverse, plane were observed. The intergroup comparison was carried out separately on each chair in both the habitual sitting and working postures. In general, changes in the sagittal curvature parameters  were found on all chairs and in both sitting positions and were most pronounced in the experienced group of dentists. No rotatory changes took place. The changes in the shoulder and pelvic parameters were not clinically relevant with regard to statistical significance. Therefore, both hypotheses 1 and hypothesis 2 have to be falsified. Despite equal gender distribution in the groups and also similar mean body heights, including standard deviations, the relevant difference in trunk length on all chairs between the groups was due to a more pronounced kyphotic thoracic spine in group 3 (experienced dentists). This could be attributed, independently of the profession, to acquired poor posture due to the older age of the subjects in group 3 –56.In the inter-chair comparison, a harmonious  sitting posture was assumed independently of the chair, both in the habitual and working postures. Only differences in trunk length and sagittal trunk decline were significant in both sitting positions. However, these significances could be attributed to a different ventral inclination of the subjects on the individual chairs, despite the positioning of the subjects with a goniometer at the beginning of measurement taking. In this context, it was found that using the chair pairs 1 and 3, 2 and 4 as well as 5 and 6 result in the most similar posture reactions during sitting. Chairs 1 and 3 were from the same manufacturer, as were chairs 5 and 6. Although chair 2 and chair 4 were based on different ergonomic sitting designs, a similar upper body statics was adopted when sitting.Overall, the differences of the inter-chair comparison could be classified as minor, since most of the statistical differences were in the range of ±1° or ± 1 mm of measurement error and, thus, a clinical relevance was negligible. Consequently, no change in lordosis and kyphosis angles could be detected when comparing the saddle chair to the other examined chairs. Therefore, hypothesis 3 has to be falsified. The results of Dable et al. , GandavaFiedler , who useIn these studies, the group of saddle chairs was found to cause a straightening of the pelvis and the associated support of the natural lumbar lordosis in comparison to conventional chairs , 58, 59,In the inter-posture comparison  the sagittal trunk decline, the thoracic bending angle and the shoulder blade distance were comparably significant in all three groups on all chairs, thus, there were no chair-specific differences. It can be confirmed that the habitual sitting posture and the working posture differ significantly in the neck and thoracal area, therefore hypothesis 4 can be verified. However, whether the working posture supports the maintenance of the natural spinal curvature cannot be assessed due to the inhomogeneity of the results of other sagittal curvature parameters. The ventral tilt of the upper body could lead to increased activity of the muscles in the lumbar region and thus favour the development of MSDs. In summary, the habitual sitting posture  Table  can be sHowever, the comparison of the two postures using videorasterstereography had some limitations due to the working posture. The ventral inclination of the upper body and the slightly ventrally inclined head meant that the marker on the vertebra prominens could not be recognized in many cases, hence post-marking was necessary. In addition, the lordosis and kyphosis angles were often calculated inadequately due to the anteinclination of the upper body which is why the results, in this regard, should be viewed critically.Furthermore, the working position adopted during the measurements was only simulated and was not examined in the actual daily work routine; the recording of the upper body statics took only a very short time and had to be carried out under laboratory conditions.Since this was a cross-sectional analysis carried out under laboratory conditions, the long-term influence of the chairs used on the sitting posture should be investigated in future studies and under real-life conditions. Studies with prolonged data cohorts in an occupational environment could establish the chair’s impacts on the occupational environment’s exposure activities in future. Despite the different ergonomic sitting layouts of the dentist’s chairs used, no significant measurable improvement in dental workplace ergonomics could be achieved. Although no measurable positive changes in upper body statics could be demonstrated, this does not mean that individual MSDs could not be reduced or prevented by one of these chairs, as discomfort is related to subjective perception. As in other studies in which different postures showed significant changes in muscle activity, joint angles, and physical activity, the results of this study have led to the conclusion that working posture has a crucial influence on the upper body posture of both dentists , 21, 38 The results showed that neither the ergonomic chair layout had any clinically relevant effect on the upper body posture, nor could a clear subjective improvement be confirmed by a particular chair. The working posture preferably adopted by the dentist may have a greater impact on the development of some musculoskeletal diseases than the chair ergonomics. The selection of the dentist’s chair contributes to a small extent  to the improvement of ergonomics in the daily dental work routine. Consequently, work related MSDs are traced back to multi-causal reasons , 23, 30.Additional file 1: Table A. Group comparison. P-values of the Kruskal-Wallis test for each chair; firstly, for the habitual sitting posture and then for the dental working posture. Table B. Chair comparison. Chi2-values of the Friedman test; first for all test persons together and then isolated for each group.


Qinghuang powder (QHP) is a traditional Chinese herbal medicine. This is a unique formula that is frequently used to treat malignant hematological diseases such as acute myeloid leukemia (AML) in modern clinical practice. An approach of network pharmacology and experimental validation were applied to investigate the pharmacological mechanisms of QHP in AML treatment. First, public databases for target genes known to be associated with AML are searched and compared to the target genes of the active compounds in QHP. Second, AML-associated genes and QHP target genes are compared to identify overlapping enriched genes, and these were used to predict selected target genes that may be implicated in the effects of QHP on AML. Additionally, we conducted functional enrichment analyses, such as gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The significantly enriched pathway associated with potential target proteins was the PI3K-Akt signaling pathway, suggesting that these potential target proteins and pathways may mediate the beneficial biological effects of QHP on AML. All these following genes were found to occur in the compounds-target-pathway networks: AKT1, MAPK1, MAPK3, PIK3CG, CASP3, CASP9, TNF, TGFB1, MAPK8, and TP53. Then, based on the molecular docking studies, it was suggested that the active compound isovitexin can fit into the binding pockets of the top candidate QHP-AML target proteins (PIK3CG). Subsequently, based on the prediction by network pharmacology analysis, both in vitro AML cells and western blot experiments were performed to validate the curative role of QHP. QHP exerted its antitumor activity on AML in vitro, as it inhibits cells proliferation, reduced the expression of Bcl-2 protein, and downregulated the PI3K-Akt signaling pathway. In conclusion, these results revealed that QHP could treat AML via a “multicomponent, multitarget, multipathway” regulatory network. Furthermore, our study also demonstrated that the combination of network pharmacology with the experimental study is effective in discovering and identifying QHP in the treatment of AML and its underlying pharmacological mechanisms. Acute myeloid leukemia (AML) is an aggressive malignancy worldwide with a poor prognosis. It is a relatively rare type of cancer , with an estimated 19, 940 new cases in the United States in 2020, according to the NIH  and SEER  databases. AML is characterized by the rapid proliferation of immature myeloid leukemia cells .Evolving pharmacological treatment strategies have led to the development of more comprehensive and multistage AML therapies, and patients are now able to use complementary and alternative medicine therapies. Traditional Chinese medicine (TCM) is a commonly used alternative medicine therapy for AML patients –12. TCM 2S2), with a small amount of arsenic trioxide (As2O3) and other heavy metal salts. QHP has been used to treat malignant hematological diseases including AML, chronic myeloid leukemia (CML), and myelodysplastic syndrome [Qinghuang powder  was used as a folk Chinese medicine, and this medicine is also described in many ancient medical books, such as Jing Yue Quan Shu, Shi Yi De Xiao Fang, and Qi Xiao Liang Fang. QHP is composed of the Chinese herbs Qing Dai  and Xiong Huang . The compounds of Indigo Naturalis include indigo, indirubin, proteins, tannic acid, and inorganic salts. The constituents of Realgar include arsenic disulfide , TCM Systematic Pharmacology Database [https://tcmspw.com/index.php), and TCM Integrated Database [http://www.megabionet.org/tcmid/) and supplemented by literature mining. The oral administration of TCM therapeutics must overcome the barriers which come in the form of absorption, distribution, metabolism, and excretion (ADME) processes to be active [All components of QHP were retrieved from the Symptom Mapping Database   and TargetNet [https://targetnet.scbdd.com/calcnet/index/) to look for targets for the components Indigo Naturalis and Realgar. These active compounds were uploaded to an online database, the species was restricted to Homo sapiens, and the target data with probability > 0 were downloaded.The proteins and gene targets of the active compounds and AML-related human genes were collected from multiple databases. Information on target proteins or genes of active compounds was collected from the TCMSP, TCMID, and SymMap databases. For comprehensiveness, we used the Swiss Target Prediction database  (https:/argetNet  (https:/https://go.drugbank.com/), DisGeNET (https://www.disgenet.org/search), GeneCards (https://www.genecards.org/), and OMIM (https://omim.org/). All protein target names were identified and converted to names of standard genes via UniProt (https://www.uniprot.org/) [https://bioinformatics.psb.ugent.be/webtools/Venn/). Finally, the QHP and AML targets that overlapped were collected using R software, and the intersection of overlapped targets was selected as the target library of this study.AML-related human genes were collected from several databases, namely, DrugBank (ot.org/) . After tThe gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the R software (version 3.6.0 for Windows). The GO enrichment analyses include biological process (BP), cellular component (CC), and molecular function (MF). The GO enrichment analysis for AML-related targets of QHP revealed remarkably 10 rich terms in BP, CC, and MF. The KEGG pathway enrichment analysis was used to elucidate the potential mechanism of QHP for AML. The bar charts and bubble diagrams were drawn with R-studio software (version 3.6.5 for Windows).https://string-db.org/) is a database of known and predicted protein-protein interactions [Biological processes involve interaction between multiple body systems are regulated by a complex regulatory network, rather than just a single gene or protein. These interactions include direct  and indirect  associations. STRING (ractions . The tarNetwork construction was achieved with Cytoscape software (ver. 3.7.1). Cytoscape is an open-source software platform for visualizing molecular interaction networks and biological pathways and then integrating these networks with annotations, gene expression profiles, and other data . Based ohttps://www.rcsb.org/) database to retrieve related protein information and downloaded the 3D structure files of the candidate proteins as the receptors. We then modified them using PyMol software (version 1.8) and AutoDockTools software (version 1.5.6) to isolate the original ligands, remove water molecules, add hydrogen, and patch amino acids. The mol2 structure of the active compounds was set to rotatable and saved as a “pdbqt” format file through the Autodock Tools software. Finally, we performed molecular docking using Autodock-vina software (version 1.1.2). The docking results were visualized with PyMol software, and the docking interaction patterns were established.To further validate the effects of QHP acting on AML, molecular docking technology was applied for screening the active compounds and candidate proteins. Molecular docking technology is an important method for the screening of drugs, which starts from known proteins and small-molecule compounds and identifies them by simulating the geometry and energy matching. We used the RCSB protein data bank (Isovitexin (lot no. abs47023192) was obtained from Absin Bioscience Inc. Roswell Park Memorial Institute (RPMI) 1640 (lot no. RHBH8770) and DMSO (lot no. RNBD8012) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. FBS (lot no. 42F7180 K) was purchased from Gibco Life Technologies Ltd. Cell Counting Kit-8 (CCK-8) kit (lot no. KGA317) was obtained from KeyGEN Biotech Co., Ltd. RIPA lysis buffer (lot no. P0013 B), PMSF (lot no. 020421210524), BeyoECL kit (lot no. P0018AS), SDS-PAGE Sample Loading Buffer 5 × (lot no. 070121210811), and BCA protein quantitation kit (Lot No. 062521210726) were purchased from Beyotime Institute of Biotechnology. BSA (lot no. EZ4567D106) was purchased from Guangzhou Saiguo Biotech Co., Ltd. Anti-PIK3CG (lot no. 5405S) were purchased from Cell Signaling Technology, Inc. Anti-AKT (lot no. CJ38131), anti-Bcl-2 (lot no. CN48171), and goat anti-mouse IgG (lot no. BS12478) were purchased from Bioworld Technology, Inc. Anti-GAPDH (lot no. AB0037) was purchased from Nanjing JinZai Biotechnology Co., Ltd. Anti-Rabbit IgG (lot no. 147832) was purchased from Jackson ImmunoResearch Laboratories, Inc.2. Once cell confluence reached∼80%, these cells were passaged.KG1-a cells were gifted from the Southern Medical University. HL-60 cells were purchased from JiangSu Rongtai Biotech Co., Ltd. Both KG1-a cells and HL-60 cells were cultured in RPMI-1640 containing 10% FBS and 1% penicillin-streptomycin at 37 °C in an incubator containing 5% COμL) and sown in Corning disposable 96-well plates for 24 h. Different concentrations of isovitexin  were incubated with KG1-a cells and HL-60 cells for 48 h. Then, the culture medium of 96-well plates was added with CCK-8 solution (culture medium: CCK-8 solution = 10 : 1). Lastly, 96-well plates were incubated at 37 °C for 1.5 h. The absorbance was read at 450 nm using Multiskan MK3 microplate reader . The half-maximal inhibitory concentration (IC50) of isovitexin was calculated using GraphPad Prism 8.3.0 software.The viability of the KG1-a cells and HL-60 cells was determined using CCK-8 kit. Firstly, KG1-a cells and HL-60 cells were suspended, respectively, in RPMI-1640 (5 × 103 cells/well in 100 μmol/L). These cells were collected from the 6-well plates followed by the addition of RIPA lysis buffer (containing 1 mM PMSF) and incubated on ice for 15 min. The recovered lysate was centrifuged at 4 °C at 14,000 ×g for 10 min. Thereafter, the protein concentration was measured with a BCA Protein Assay Kit. An equal amount of protein (50 μg/lane) was separated by 12 SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore). Subsequently, the PVDF membranes were blocked with 5% skim milk for 1 h and incubated overnight with different primary antibodies against PIK3CG , AKT (1 : 500 dilution), Bcl-2 (1 : 500 dilution), and GAPDH , respectively, at 4 °C. Following 3 washes, the membranes were incubated for 1 h with secondary horseradish peroxidase-conjugated antibody at room temperature. Immunolabeled protein bands were detected with ECL substrate. Semiquantitative analysis was performed using ImageJ software. Target protein levels were normalized against the level of GAPDH.Further to study how isovitexin affects these cell signaling pathways, KG1-a cells and HL-60 cells were plated in 6-well plates (1 × 104 cell/well) and treated for 48 h with a variety of isovitexin concentrations . Statistical significance was set at p value < 0.05.Data were expressed as mean and standard deviation (SD) from at least three independent experiments. The statistical significance of differences between the two groups was determined using Student's 2O3, As, AsS, and As4S4) do not meet the ADME parameters and were therefore excluded.The QHP formula contains two Chinese medicinal constituents. They are Indigo Naturalis and Realgar. We identified a total of 48 compounds in QHP through the TCMSP, TCMID, and SymMap databases, 38 of which were contained in Indigo Naturalis and 10 in Realgar. Among the 38 compounds in Indigo Naturalis, 8 (26.3%) met the requirement of OB ≥ 30% and DL ≥ 0.18 based on the TCMSP database, and the 4 compounds in Realgar also met this criterion based on the TCMID and SymMap databases. Therefore, 14 compounds were chosen as candidate active compounds for further analysis. A detailed list of these compounds is shown in Among the 14 active compounds, 319 target genes were retrieved from the TCMSP, TCMID, TargetNet, and SymMap databases. After eliminating duplicates, 261 gene targets were obtained for further study. In total, 947 AML-related human gene targets were collected from OMIM, GeneCards, DisGenet, DrugBank, and SymMap databases. We then extracted all the unique gene targets from each result. The intersection of these two types of gene targets was then collected for further analysis . As a re4S4 (degree = 60), beta-sitosterol , As2O3 (degree = 46), indirubin , AsS (degree = 23), 5-O-methylvisamminol , indican , indigo , isoindigo , isovitexin , qingdainone , and quindoline . These high-degree compounds in the network were deemed to relate to the essential therapeutic effects of QHP on AML.The targets of 13 compounds that were associated with AML were imported into Cytoscape (ver. 3.7.1), and the QHP-AML target network diagram that was constructed is shown in https://www.string-db.org/), and the results were downloaded as a “TSV” file. The TSV file was imported into Cytoscape (ver. 3.7.1), and the diagram of the gene target network was constructed as shown in The 89 gene targets shared between QHP and AML were imported into the STRING database , cellular component (CC), and molecular function (MF). The top 10 significantly enriched GO terms in BP, CC, and MF of the potential targets are shown in Based on the above GO terms and KEGG pathway enrichment analyses, the compounds-target-pathway network was generated with Cytoscape (version 3.7.2), which connected these compounds, potential targets, and corresponding pathways. The compounds-target-pathway network is shown in In order to further validate the ability of the active compounds in QHP to bind with the key targets, molecular docking visualization techniques were used to examine systematic docking. We chose to research these underlying target proteins in the C-T-P network because they were high-degree nodes with multifunctional connections. The top 10 key targets based on degree were as follows: AKT1, MAPK1, MAPK3, PIK3CG, CASP3, CASP9, TNF, TGFB1, MAPK8, and TP53. A total of 13 compounds were selected for docking on the 10 target proteins under the procedure. The thermal map of the lowest binding energy is shown in −1) was below −7.0, indicating good binding activity between the compound and the protein [a, b, and c. In our schema, “a” shows the binding position of the small-molecule compound (sticks model) on the associated protein (cartoon model) as a whole; “b” shows the interaction between the small molecule (green) and the key residue (grey) on the protein of interest, which facilitates the observation of the spatial stacking between the compound and the protein; and “c” shows a two-dimensional view of the hydrogen bonding and hydrophobic (stacking) interactions between the compound and protein residues.Docking analysis successfully predicted docking between QHP and the binding pockets of 10 target proteins. The most active compound in QHP based on docking results was isovitexin. The value of binding affinity  and 1.258 μmol/L (HL-60 cells). These results suggest that isovitexin can inhibit AML cell growth.In comparison to the control group, different concentrations of isovitexin had inhibitory effects on KG1-a cells and HL-60 cells in a dose-dependent manner. As shown in μmol/L), the protein levels of PIK3CG, AKT, and BCL-2 in KG1-a cells and HL-60 cells were decreased in a dose-dependent manner . Taken together with the results of cell viability measurement, the anti-acute myeloid leukemia activities of isovitexin seem to be mediated by inhibition of the cell growth and regulation of the PI3K-AKT signaling pathway.Based on the “3.5.1 GO and KEGG” pathways analyzed and “3.6 Molecular Docking” docking results, the PI3K-AKT signaling pathway was selected for experimental validation as a major tumor-related signaling pathway. As shown in AML is a highly complex cancer in terms of its molecular and cytogenetic architecture, which are involved with multiple genes or signaling pathways during the development and progression of the disease . Because2O3 and As4S4 that could be retrieved from the TCMSP database including OB and DL values. However, there is increasing evidence to associate both As2O3 and As4S4 with the AML pathological process [4S4 and As2O3 are the primary bioactive compounds in QHP [G0 arrest and PML-RARα degradation [Some compounds did not exhibit pharmacokinetic properties that could allow them to be delivered to target organs to generate biological activities. In modern integrated drug development, these compounds with OB ≥ 30% and DL ≥ 0.18 were seen as good pharmacokinetically active compounds . There i process –39. Mores in QHP . In thiss in QHP , 42. Morradation , 44. MeaIn the process of anticancer drug discovery, the search for key targets that are linked together is a major focus of research. One drug can be linked to many genes and proteins in complex relationships. Similarly, one gene or protein can be linked to many drugs. At the same time, there are more and more online analysis tools that can be used in network pharmacology studies. In this study, 89 potential target genes related to QHP and AML were identified with TCMSP, TCMID, SymMap, OMIM, GeneCards, DisGenet, and Drugbank database analysis. After that, 89 potential target genes were analyzed based on the STRING database. In order to comprehensively understand their biofunctional annotation information, GO terms, and KEGG pathway enrichments, they were analyzed with R language. According to the analysis of BP terms, QHP is closely related to aging, peptidyl-serine modification, peptidyl-serine phosphorylation, protein autophosphorylation, reactive oxygen species metabolic processes, and so on. These results may be related to targeting AML effects of QHP , 46. Wha4S4 and As2O3 are small. Despite their high degree, the binding effects of As4S4 and As2O3 with proteins are not as strong as that of macromolecules. Moreover, it is significant that these target genes with high-degree are closely related to the PI3K-Akt signaling pathway. Biological studies showed that the dysregulation of the PI3K-Akt signaling pathway in AML may cause the activation of downstream signal molecules, regulate tumor cell proliferation, apoptosis processes, and mediate tumor cell resistance to radiotherapy and chemotherapy [In addition, we made a C-T-P network diagram. In this network, nodes with high-degree may be considered to represent the major therapeutic effects of QHP on AML. Compared to macromolecular compounds, the molecular weights of Asotherapy , 48. Dysotherapy , 50. SimIn this study, network pharmacology techniques were used to elucidate the potential antileukemia effects of QHP, and these effects were visualized by molecular docking for verification. This systematic theoretical research provides a fundamental methodology for future pharmacological research designed to further explore the mechanism of action of QHP in the treatment of AML. However, this study has some shortcomings that require further research. Firstly, the public web database selected for this study is characterized by real-time updates, so the results of this study can only partially elucidate the molecular mechanism of QHP against AML. Secondly, only molecular docking and in vitro experimental validation were performed in this study, and a multidimensional validation should be performed subsequently in combination with animal models and clinical trials. Thirdly, studies on the quantitative determination of the content of 14 compounds in QHP are currently not yet perfect. Moreover, isovitexin, though determined as the mainly important bioactive compound of QHP against AML, could not completely stand for QHP. Thus, additional study is required to further explore the underlying molecular mechanism of QHP in the treatment of AML in vitro and in vivo.Based on the network pharmacology, molecular docking analysis, and experimental validation, the underlying mechanism of QHP in AML therapy involves the regulation of signaling pathways and targets. In this study, we identified that isovitexin might be the underlying compounds responsible for the therapeutic actions of QHP. The anti-AML effects of QHP seem to be mediated by inhibition of the AML cells growth and Bcl-2 protein expression, and regulation of the PI3K-Akt signaling pathway. Overall, the obtained results suggested that QHP might be used as a promising therapeutic agent for AML and provide additional evidence for the promotion of the wide use of QHP in the clinic for the treatment of AML diseases.


SGCE gene encoding ε-sarcoglycan leading to a dysfunction of this transmembrane protein, alterations in the cerebello-thalamic pathway and impaired striatal plasticity. To elucidate underlying pathogenic mechanisms, we investigated induced pluripotent stem cell (iPSC)-derived striatal medium spiny neurons (MSNs) from two myoclonus-dystonia patients carrying a heterozygous mutation in the SGCE gene (c.298T>G and c.304C>T with protein changes W100G and R102X) in comparison to two matched healthy control lines. Calcium imaging showed significantly elevated basal intracellular Ca2+ content and lower frequency of spontaneous Ca2+ signals in SGCE MSNs. Blocking of voltage-gated Ca2+ channels by verapamil was less efficient in suppressing KCl-induced Ca2+ peaks of SGCE MSNs. Ca2+ amplitudes upon glycine and acetylcholine applications were increased in SGCE MSNs, but not after GABA or glutamate applications. Expression of voltage-gated Ca2+ channels and most ionotropic receptor subunits was not altered. SGCE MSNs showed significantly reduced GABAergic synaptic density. Whole-cell patch-clamp recordings displayed elevated amplitudes of miniature postsynaptic currents and action potentials in SGCE MSNs. Our data contribute to a better understanding of the pathophysiology and the development of novel therapeutic strategies for myoclonus-dystonia.Myoclonus-dystonia  is characterized by alcohol-sensitive, myoclonic-like appearance of fast dystonic movements. It is caused by mutations in the  Myoclonus-dystonia  with onset in childhood or adolescence is characterized by a combination of myoclonic jerks and dystonic symptoms ,3,4. TheThis movement disorder can only be treated symptomatically with systemic pharmacological therapy, e.g., anticholinergic or GABAergic drugs, intramuscular botulinum toxin injection, or deep brain stimulation ,12,13. Tε-sarcoglycan gene (SGCE) iodobenzamide (IBZM)) study of SGCE mutation carriers, a decreased dopamine D2-receptor availability in the striatum could be detected i). For analysis of spontaneous Ca2+ transients, only Ca2+ signals with a 340/380 nm excitation ratio of Fura 2-AM of ≥ 0.02 of individual MSNs were used. For analysis of response amplitudes after neurotransmitter application, only Ca2+ signals with a 340/380 nm excitation ratio of Fura 2-AM ≥ 0.05 of individual MSNs were used . Gene expression of three independent differentiations per cell line (two control lines and two SGCE lines), each in triplicate, were used for analysis and illustrated as means ± standard error of the mean (SEM). For a list of all primer sequences of GABAA receptor [For RNA extraction, iPSCs and differentiated MSNs (collected at day 70 days ± 7 days) were processed using RNeasy Mini Kit  including a column DNase digestion. The quality of total RNA was checked by Nanodrop analysis . A total of 250 ng of RNA was transcribed into cDNA using QuantiTect reverse transcription kit (Qiagen). Quantitative real-time PCR reaction analysis was performed  using Poreceptor , voltage2, 10 mM HEPES, and 5 mM EGTA, adjusted to pH 7.3 with KOH, 305 mOsm). The external bath solution contained 142 mM NaCl, 8 mM KCl, 1 mM CaCl2, 6 mM MgCl2, 10 mM glucose, and 10 mM HEPES, adjusted to pH 7.4 with NaOH, 325 mOsm). Whole-cell currents were low-pass filtered at 2.9 kHz, digitized at 10 kHz using an EPC-10 amplifier , and analyzed with PatchMaster and FitMaster software (HEKA). Sodium and potassium ion currents were elicited by depolarizing voltage steps in increments of 10 mV from a holding potential of −70 to 40 mV. Miniature postsynaptic currents (mPSCs), indicating mostly GABAergic synaptic activity in MSNs [Electrophysiological patch-clamp measurements of MSNs were performed after 70 days ± 7 days of differentiation in vitro from at least three to four independent differentiations per iPSC line as described previously ,59,64. T in MSNs ,38, werep-value) was set to p < 0.05 with * p < 0.05, ** p < 0.01, *** p < 0.001.Statistical analysis was performed using GraphPad Prism 5 Software . All data of MSNs from healthy controls and SGCE lines were pooled in control and SGCE groups for the respective experiments and are presented as mean ± standard error of the mean (SEM). When the two groups showed normal distribution, a two-tailed unpaired t-test, in other cases a nonparametric Mann–Whitney-U test was calculated comparing the two groups (control versus SGCE). Two-way ANOVA followed by Bonferroni post hoc analysis was used for multiple comparisons, when comparing more than two groups. The significance level (


Hymenoptera stings mostly cause acute urticaria but we describe a case of CU after wasp stings which remitted during venom immunotherapy. IgE‐mechanisms have not been fully clarified in CU, except for isolated circumstances. In our case immunotherapy has played a positive role reducing immune cells reactivity and improving urticaria symptoms. Hymenoptera stings mostly cause acute urticaria but we describe a case of CU after wasp stings which remitted during venom immunotherapy. IgE‐mechanisms have not been fully clarified in CU, except for isolated circumstances. In our case immunotherapy has played a positive role reducing immune cells reactivity and improving urticaria symptoms. Hymenoptera sting causes acute urticaria and no report of CU after Hymenoptera sting can be found in the literature. We describe a case of onset of CU after multiple wasp stings that remitted during venom immunotherapy.Urticaria is a clinical condition characterized by the development of angioedema, wheals (hives), or both. Urticaria affects about 15%‐20% of the population once or more during a lifetime, and 0.5%‐1.0% suffer from CU. Urticaria classification is based on its duration: Acute urticaria is distinct by the spontaneous occurrence of wheals, angioedema, or both for less than 6 weeks, while chronic urticaria (CU) is defined as the appearance of the symptoms for more than 6 weeks. CU is classified in different subtypes: chronic spontaneous urticaria (CSU) and inducible urticaria, with a very wide spectrum of clinical manifestations between these subtypes. Additionally, two or more different subtypes of urticaria can coexist in any given patient. Inducible urticaria comprises symptomatic dermographism, cold urticaria, delayed pressure urticaria, solar urticaria, heat urticaria, vibratory angioedema, cholinergic urticaria, contact urticaria, and aquagenic urticaria. CSU can also be due to unknown or known causes, for example, autoreactivity, that is, the presence of mast cell‐activating autoantibodies.Helicobacter pylori or Anisakis simplex) have been described as causes of CU.Urticaria is a disease driven by mast cells: Activated dermal mast cells release histamine, platelet‐activating factor, other mediators, and cytokines, but the pathogenesis is complex and has many additional features. In urticaria, the mast cell‐activating signals are heterogeneous and ill‐defined. There is a long list of both exogenous and internal causes of urticaria, including autoimmunity, infections, and allergic disorders. In the past decades, many advances have been made in identifying causes of different types and subtypes of urticaria. Among others, autoimmunity mediated by functional autoantibodies directed against the high‐affinity IgE receptor or IgE‐autoantibodies to auto‐antigens, pseudo‐allergy  to foods or drugs, and acute or chronic infections , after months of unsuccessful therapy for CU.Helicobacter pylori fecal antigen and beta‐hemolytic streptococci from throat and nasal swabs. Subtle urinary tract infection, including Chlamydia spp. and Mycoplasma spp., was excluded. Acute or misunderstood chronic viral infections, such as hepatitis , Epstein‐Barr virus, cytomegalovirus, and other herpesviruses, were investigated. Antibodies against thyroid peroxidase, thyroid‐stimulating hormone receptor, anti‐thyroglobulin, anti‐endomysial, and anti‐tissue transglutaminase resulted negative. Plasma levels of D‐Dimer and acute phase reactants  were normal. The levels of complement components excluded complement consumption or deficiencies. Autologous serum skin test excluded the presence of functional autoantibodies. Skin tests with common inhalant and food allergens (Lofarma), Apis mellifera , and Polistes dominula  venoms were negative. Vespinae venoms tested positive, both Vespula spp (ALK‐Abelló) and Vespa crabro , at concentration of 0.1 μg/mL. Specific IgE tested positive for vespid venoms, respectively, 5.46 kU/L for Vespula spp, 0.47 kU/L V crabro, 0.69 kU/L P dominula. Analysis of the venom‐specific component revealed rVes v 5 (6.17 kU/L), rVes v 1 (< 0.10 kU/L), and rPol d 5 (1.07 kU/L). Total IgE level was 132 kU/L and serum baseline tryptase 7 mcg/mL. Notably, after skin tests with vespid venoms the patient suffered from an exacerbation of urticaria on his hips and thighs. Baseline assessment of disease activity with the urticaria activity score (UAS) showed a scarce control with oral antihistamine. Then, he started oral prednisone 25 mg/day, reaching the daily lowest effective dose of 5 mg. After 3 months, CU symptoms partially improved, and he agreed to start VIT because of the impairment of his quality of life and the close beginning of wasp season. Venom‐specific immunotherapy was started with Vespula spp extract (ALK‐Abelló) according to a 6‐week cluster schedule with weekly incremental doses of venom extract subcutaneously, until a maintenance dose of 100 000 standard quality units (SQ‐U/mL) was reached. During the up‐dosing phase, the patient remained under antihistamines therapy, because urticaria was still present. The achieved maintenance dose (100 000 SQ‐U/mL) was then given every 4‐6 weeks. During the up‐dosing phase and maintenance treatment, minor side effects such as local flushing and mild itching were reported and tolerated, and a worsening of the urticarial lesions occurred the day after each VIT injections. Nevertheless, after few months of VIT both pruritus and wheals improved gradually, and after 10 months, urticaria suddenly remitted and antihistamines were stopped. At present, CU symptoms have not recurred and the patient continues VIT .He had a negative history of allergic and autoimmune diseases, and previous anaphylactic episodes, only a familiar history of intolerance to Aspirin and a personal history of gastroesophageal reflux disease, not requiring any therapy at that moment. He referred the first sting at the age of 20 by an unidentified insect and experienced a normal local reaction . Afterward, about 1 year before our examination, he was stung by a vespid and again experienced a normal local reaction. Two months later, about 20 minutes after receiving multiple stings by a wasp swarm, itching hives developed all over his body. At the local hospital, intravenous methylprednisolone acetate and intramuscular chlorpheniramine were administered, with complete regression in 24 hours, and oral antihistamine for a week was prescribed. The day after, though, he manifested satellite lymphadenitis, which regressed in 5 days with steroid cream. Three weeks later, migrant generalized urticarial lesions started, showing spontaneous short‐lived wheals not in relation to foods or drugs, physical agents, or exercise. At our facility, routine blood tests were normal, except for mild leucopenia, which was already known before urticaria occurred; basophil and eosinophil counts were normal. Other causes of CU, such as acute or chronic infections and thyroid gland disorders, were excluded. Because of the prevalence of infections in our region, we ruled out the presence of 3The diagnostic workup of CU includes among the major aims to exclude differential diagnoses.Our patient never showed angioedema. This main feature allowed us to exclude diseases and syndromes defined by episodes of angioedema without hives, such as nonmast cell mediator‐mediated angioedema  and Gleich's syndrome (episodic angioedema with eosinophilia).To distinguish different pathophysiologic mechanisms underneath the appearance of wheals is important to observe their cutaneous characteristics, their duration, and the presence of itching or burning sensation. As in our case, wheal in patients with urticaria has three typical features: a central swelling surrounded by reflex erythema, a fleeting nature lasting less than 24 hours, and association with itch. Therefore, we excluded the maculopapular cutaneous mastocytosis (urticaria pigmentosa) because of its peculiar dark‐colored skin lesions with a duration more than 24 hours, and the prebullous stage of bullous pemphigoid because of the absence of the typical vesicular eruption. In patients who display only wheals (but no angioedema), urticarial vasculitis, and auto‐inflammatory disorders, such as Schnitzler's syndrome or cryopyrin‐associated periodic syndromes (CAPS), need to be ruled out. Hives of autoimmune and auto‐inflammatory disorders have the typical feature of lasting more than 24 hours. Additionally, besides urticarial rash, CAPS  are characterized by recurrent fever attacks, arthralgia or arthritis, eye inflammation, fatigue and headaches, and Schnitzler's syndrome is characterized by recurrent fever attacks, bone and muscle pain, arthralgia or arthritis and lymphadenopathy and monoclonal gammopathy.Serum sickness and serum sickness‐like reactions have been suspected because they can be triggered by insect stings and present with pruritic urticarial‐type lesions and lymphadenopathy without involvement of the mucous membranes. Symptoms appear days after the sting, overlapping a delayed reaction. We excluded these disorders because, unlike urticaria, the lesions often persist for days in the same area, and arthralgia or arthritis and fever are associated. Additionally, the long‐lasting skin lesions and the little improvement with steroid therapy are featured more typical of CU rather than serum sickness and serum sickness‐like reactions.The hypothesis behind urticaria progression in our case can be explained from several perspectives. First, vespid sting and urticaria onset could be coincidental and CU remitted spontaneously with VIT exerting no effect on its course. In fact, in most cases, CU is a self‐limiting disorder, persisting for 2‐5 years.Alternatively, CU progression, from onset to remission, might be associated with some specific effects of venom allergens, from sting to VIT. Supporting this hypothesis, we observed a clear‐cut association between exposure to venom allergens and urticaria symptoms. In fact, type I allergy may be causative in a small number of CU patients and specific immunotherapy with these allergens may be beneficial in those patients.Moreover, besides its specific effects, AIT exerts some nonspecific effects. AIT changes basophil and mast cell homeostasis and reduces the skin sensitivity not only to specific allergens, but also to histamine and nonspecific mast cell stimuli. Immunologic changes induced during the course of AIT include reduced mast cell and basophil activity and degranulation. After a few months, AIT produces a decrease in tissue mast cell, both connective and mucosal, in innate type 2 lymphocytes and eosinophil counts as well as in the release of their mediators.4This is the first report in the literature of onset of chronic urticaria after Hymenoptera sting. Due to the need to undertake immunotherapy to protect the patient from venom allergy, a clear‐cut association between exposure to venom allergens and urticaria symptoms was revealed. In fact, IgE‐mediated allergy is a rare cause of CU, but should be considered in patients with intermittent symptoms. Additionally, besides its specific effects, immunotherapy exerts nonspecific effects on basophil and mast cell homeostasis. In our case, CU progression, from onset to remission, might be associated with some specific effects of venom allergens, from sting to allergen‐specific immunotherapy. This is the first report in the literature of a remission of chronic urticaria during venom immunotherapy.The authors certify that there is no conflict interest with any financial organization regarding the material discussed in the manuscript.VP and FR: provided the patient data regarding the allergic disease; MD and VP; wrote the manuscript; RA and GS: made contributions to interpretation of data. All authors critically read and approved the final manuscript.All procedures were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2013. Informed written consent was obtained from the patient.


We have previously shown that exercise training prevents arrhythmias in CPVT1, potentially by reducing the oxidation of Ca2+/calmodulin‐dependent protein kinase type II (CaMKII). Therefore, we tested whether an oxidation‐resistant form of CaMKII protects mice carrying the CPVT1‐causative mutation RyR2‐R2474S (RyR2‐RS) against arrhythmias. Antioxidant treatment (N‐acetyl‐L‐cysteine) reduced the frequency of β‐adrenoceptor stimulation‐induced arrhythmogenic Ca2+ waves in isolated cardiomyocytes from RyR2‐RS mice. To test whether the prevention of CaMKII oxidation exerts an antiarrhythmic effect, mice expressing the oxidation‐resistant CaMKII‐MM281/282VV variant (MMVV) were crossed with RyR2‐RS mice to create a double transgenic model (RyR2‐RS/MMVV). Wild‐type mice served as controls. Telemetric ECG surveillance revealed an increased incidence of ventricular tachycardia and an increased arrhythmia score in both RyR2‐RS and RyR2‐RS/MMVV compared to wild‐type mice, both following a β‐adrenoceptor challenge , and following treadmill exercise combined with a β‐adrenoceptor challenge. There were no differences in the incidence of arrhythmias between RyR2‐RS and RyR2‐RS/MMVV mice. Furthermore, no differences were observed in β‐adrenoceptor stimulation‐induced Ca2+ waves in RyR2‐RS/MMVV compared to RyR2‐RS. In conclusion, antioxidant treatment reduces β‐adrenoceptor stimulation‐induced Ca2+ waves in RyR2‐RS cardiomyocytes. However, oxidation‐resistant CaMKII‐MM281/282VV does not protect RyR2‐RS mice from β‐adrenoceptor stimulation‐induced Ca2+ waves or arrhythmias. Hence, alternative oxidation‐sensitive targets need to be considered to explain the beneficial effect of antioxidant treatment on Ca2+ waves in cardiomyocytes from RyR2‐RS mice.Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited arrhythmogenic disorder caused by missense mutations in the cardiac ryanodine receptors (RyR2), that result in increased β‐adrenoceptor stimulation‐induced diastolic Ca 2+/calmodulin‐dependent protein kinase type II (CaMKII) prevents arrhythmias in murine models of catecholaminergic polymorphic ventricular tachycardia (CPVT). CaMKII can be activated by the oxidation of methionines 281/282, and we show that antioxidant treatment may limit arrhythmogenic activity in CPVT. However, genetic inhibition of CaMKII oxidation at methionines 281/282 does not protect CPVT mice from arrhythmias. Other oxidation‐sensitive targets should be explored to explain the effect of antioxidants in CPVT.Inhibition of Ca If untreated, CPVT has a mortality rate of 30%–50% by the age of 40   is an inherited disorder characterized by β‐adrenoceptor stimulation‐induced ventricular tachycardia (VT)  , which conforms to the European Union Directive for the protection of experimental animals 2010/63/EU. Animals were housed in cages with a 12 h light/dark cycle and had ad libitum access to food and water.2.2Mice with the knock‐in CaMKIIδ‐MM281/282VV mutation (MMVV) with M281 and M282 are replaced with valines to prevent oxidation  was implanted subcutaneously in the lumbar back of the mouse. The ECG electrodes were placed subcutaneously on each lateral side of the thoracic wall. One week was allowed for recovery from the surgery before ECG recordings. A stress test was performed using i.p. administration of 20 mg/kg isoprenaline . One week was allowed for recovery from the stress test before a second stress test was conducted consisting of treadmill running followed by isoprenaline administration. Before the second stress test, mice were acclimatized to the treadmill (Columbus Instruments) for 15 min per day for three consecutive days at low speeds . The stress test was initiated with a speed of 3.6 m/min and increased stepwise by 1.8 m/min every 1.5 min until the mice were unable to continue running (defined as >1 s off the treadmill belt). Isoprenaline was administered promptly after the discontinuation of running. Arrhythmias occurring during the first five minutes following isoprenaline administration were analyzed using Ponemah software . An arrhythmia score based on the most severe arrhythmia was applied: no arrhythmias = 0; isolated ventricular extrasystoles (VES) = 1; VES in bigeminy = 2; couplets = 3; VT = 4. VT was defined as three or more consecutive VES. Investigators were blinded to the animal genotype during both experiments and analyses.2.42, 0.4 NaH2PO4; pH adjusted to 7.4 with NaOH). The heart was then mounted on a modified Langendorff setup and perfused with isolation buffer at 37℃. The perfusion solution was gradually switched to an isolation buffer containing 0.012 mM Ca2+ and ~500 U/mL of collagenase type II (Worthington). Following digestion for 6–10 min, the left ventricle was isolated, transferred into isolation buffer with 0.02 mM Ca2+ and 0.1% bovine serum albumin, cut into pieces, and triturated with ~100 U/mL of deoxyribonuclease I (Worthington). The solution was filtered through a Nitex mesh with a pore size of 255 µm and the Ca2+ concentration increased stepwise every ~4 min to 0.05, 0.1, 0.2, and 0.5 mM. Cardiomyocytes were kept at room temperature and used within 6 h.Before anesthesia induction, ~50 IU heparin/20 g i.p. was administered. Anesthesia induction and maintenance were conducted in the same manner as described under Telemetric ECG surveillance. Following thoracotomy, the heart was excised and put in ice‐cold isolation buffer  for 15 min including 5 min on laminin‐coated coverslips mounted in a perfusion chamber. Cardiomyocytes were superfused with a modified HEPES‐buffered Tyrode's solution  at 37 ± 0.5℃. A Zeiss Axio Observer microscope with a PTI D‐104 microscope photometer and PTI Felix software were applied to record whole‐cell Ca2+ fluorescence intensity. Rod‐shaped cardiomyocytes without membrane blebs were paced using platinum electrodes at ~20% above individual threshold for contraction at either 1 or 4 Hz for 20 s. Ca2+ waves were studied in a 10 s post‐pacing rest period. If no Ca2+ waves occurred in the rest period, Ca2+ wave latency was set to 10 s. Sarcoplasmic reticulum (SR) Ca2+ content was estimated from the Ca2+ release evoked by the application of 10 mM caffeine (Sigma Aldrich) following 20 s of 1 Hz pacing. The effects of β‐adrenoceptor stimulation were assessed following the application of 100 nM isoprenaline  for a minimum of 100 s. Ca2+ reuptake rate was measured as the rate constant of the Ca2+ transient decay (1/tau) using ClampFit software . SR Ca2+ ATPase (SERCA)‐dependent Ca2+ reuptake was calculated by subtracting the rate constant of the caffeine transient from the rate constant of the Ca2+ transient. To study the effect of antioxidant treatment on Ca2+ waves in RyR2‐RS, cardiomyocytes were preincubated with 5 mM N‐acetyl‐L‐cysteine (NAC) for 20 min prior to superfusion with the HEPES‐buffered Tyrode's solution with 5 mM NAC added. Investigators were blinded to the treatment with NAC (vs. vehicle), or to the animal genotype, during both experiments and analyses.Cardiomyocytes were incubated with 2 µM of the Ca2.6Anesthesia was induced and maintained as described under Telemetric ECG surveillance. A thoracotomy was performed and the heart was excised and put in ice‐cold saline. To study the effects of β‐adrenoceptor stimulation on phosphoprotein status 20 mg/kg isoprenaline  was administered i.p. 3 min before excision of the heart in a cohort of mice. The left ventricle was isolated, snap‐frozen in liquid nitrogen, and stored at −80℃. The frozen tissue was homogenized in lysis buffer  using a Polytron 1200 homogenizer. Phosphatase and protease inhibitor cocktails (Roche Diagnostics) were added. Micro BCA protein assay kit (Thermo Fisher Scientific) was used to quantify protein concentrations. Sample buffer  was added to the protein extract followed by heating at 94℃ for 5 min. The samples were size fractioned on Criterion 4–15% Tris–HCl gels (15% for phospholamban) and transferred to 0.2 µm PVDF‐membranes (Bio‐Rad Laboratories). Blots were blocked in 3% BSA or 5% non‐fat milk in Tris‐buffered saline with 1% Tween and incubated with primary antibodies overnight and secondary antibodies for one hour at room temperature. The following antibodies were used: anti‐CaMKII phospho T286 , anti‐RyR2 phospho S2808 (A010‐30), anti‐RyR2 phospho S2814 (A010‐31), anti‐phospholamban phospho S16 (A010‐12), anti‐phospholamban phospho T17 (A010‐13) , anti‐phospholamban (MA3‐922), anti‐CaMKIIδ (PA5‐22168), anti‐RyR2 (MA3‐916) (Thermo Fisher Scientific Inc.), anti‐GAPDH , anti‐vinculin . Anti‐goat, anti‐rabbit or anti‐mouse IgG horseradish peroxidase‐conjugated antibody  was used as the secondary antibody. Amersham ECL Prime  was used to develop blots and signals were detected by LAS‐4000 (Fujifilm Life Science).2.72+ wave frequency. Fisher's exact test was used to study the proportion of animals developing VT. Kruskal–Wallis test was used to compare arrhythmia scores and arrhythmia events. Unpaired t‐test was used for comparison of phosphoproteins.Results are reported as the mean ± standard error of the mean. A Nested ANOVA using the lme function in R software for statistical programming was used for comparisons of all data from isolated cardiomyocytes . These data indicate that the inhibition of oxidation by antioxidant treatment may protect against arrhythmogenic Ca2+ release in CPVT1.We tested whether antioxidant treatment could prevent arrhythmogenic Cas Figure . Under bs Figure . Furthers Figure . Arrhythy Figure . NAC didn Figure . However3.2Next, we specifically tested whether preventing CaMKII oxidation on the MM281/282 oxidation site could protect CPVT1 mice from β‐adrenoceptor stimulation‐induced arrhythmias. For this purpose, arrhythmias were studied in RyR2‐RS/MMVV, RyR2‐RS, and WT mice following i.p. administration of 20 mg/kg isoprenaline Figure . As expe3.32+ waves in isolated cardiomyocytes  and in mice exposed to i.p. administration of isoprenaline three min before harvest to examine the effects of β‐adrenoceptor stimulation Figure . RyR2‐RS42+ waves in CPVT1.CaMKII is a promising target for arrhythmia prevention in CPVT. Increased understanding of how CaMKII contributes to the arrhythmogenic phenotype in CPVT can help bring this strategy closer to clinical use. Here, we find that the oxidation‐resistant CaMKII MM281/282VV mutation does not prevent arrhythmias or arrhythmogenic Ca4.12+ release through the oxidation of CaMKII. We have previously shown that exercise training reduces CaMKII oxidation, CaMKII‐dependent RyR2‐S2814 phosphorylation, arrhythmogenic Ca2+ release, and arrhythmias in the RyR2‐RS CPVT1 mouse model . However, the small differences in Ca2+ wave frequency are comparable to previous reports (Bonilla et al., 2+ waves, Ca2+ sparks and “invisible” leak also contribute to the total Ca2+ leak (Hoang‐Trong et al., 2+ leak mediated by Ca2+ sparks is substantially higher in RyR2‐RS than WT (Sadredini et al., 2+ waves during pacing may better reflect the physiological situation (Fernández‐Velasco et al., One surprising observation in our study is that we only find increased Ca4.32+ transient amplitude in both RyR2‐RS and RyR2‐RS/MMVV compared to WT despite similar SR Ca2+ content. This is presumably due to increased fractional Ca2+ release caused by increased opening probability in RyR2‐RS (Lascano et al., 2+ transient amplitude is unaltered in both RyR2‐RS and RyR2‐RS/MMVV at 1 Hz pacing compared to WT and even reduced at 4 Hz pacing in RyR2‐RS compared to WT. The latter is consistent with previous works and often ascribed to reduced SR Ca2+ content in CPVT (Fernández‐Velasco et al., 2+ content following 1 Hz pacing. However, the above‐mentioned rate‐dependent SR Ca2+ depletion in CPVT1 (Fernández‐Velasco et al., 2+ transient amplitude in RyR2‐RS at 4 Hz pacing. Surprisingly, the Ca2+ transient amplitude was increased in RyR2‐RS/MMVV compared to RyR2‐RS at 1 Hz pacing during β‐adrenoceptor stimulation. We did not detect any significant difference in SR Ca2+ content between RyR2‐RS/MMVV and RyR2‐RS. We can only speculate whether minor effects of the MM281/282VV mutation on CaMKII targets, such as the L‐type Ca2+ channel, have contributed to the increased Ca2+ transient amplitude.Under baseline conditions during 1 Hz pacing, we find increased Ca52+ release or arrhythmias in CPVT1. Concurrently, we find antioxidant treatment to reduce arrhythmogenic Ca2+ release in CPVT1. Future studies are needed to further examine the role of other targets for oxidation to advance new therapeutic strategies for CPVT.Our data demonstrate that genetically engineered resistance to CaMKII MM281/282 oxidation does not protect against arrhythmogenic CaNone declared.All authors conceived and designed research; M.S. and R.M. performed experiments; M.S. analyzed data; all authors interpreted results of experiments; M.S. and M.K.S. drafted the manuscript; all authors edited and revised the manuscript; all authors approved the final version of the manuscript.