Acute myeloid leukemia (AML) is rare in children. Although complex karyotype (CK) defined as ≥ 3 cytogenetic abnormalities is an adverse risk factor in adult AML, its prognostic impact on childhood AML remains to be determined.p = 0.053) and event-free survival (EFS)  compared with those with intermediate-risk genetics. Typical CK tended to correlate with a decreased OS than atypical CK , and CK with ≥ 5 cytogenetic aberrations was associated with an inferior survival compared with CK with ≤ 4 aberrations .We studied the prevalence, cytogenetic and mutational features, and outcome impact of CK in a cohort of 284 Chinese children with de novo AML. Thirty-four (12.0%) children met the criteria for CK-AML with atypical CK being more frequent than typical CK featured with -5/5q-, -7/7q-, and/or 17p aberration. Mutational prevalence was low and co-occurrence mutants were uncommon. Children with CK-AML showed shorter overall survival (OS) (5-year OS: 26.7 ± 10.6% vs. 37.5 ± 8.6%, Our results demonstrate CK as an adverse risk factor for reduced survival in childhood AML. Our findings shed light on the cytogenetic and mutational profile of childhood CK-AML and would inform refinement of risk stratification in childhood AML to improve outcomes.The online version contains supplementary material available at 10.1186/s13039-021-00547-0. Acute myeloid leukemia (AML) is a group of clonal hematopoietic neoplasms that are characterized by aberrations in maturation, proliferation, and survival in the stem and progenitor cell compartments. Childhood AML is a relatively rare disease that accounts for 15%–20% of acute leukemias in children. Despite considerable progress have made in treating children with AML, about 30% of patients still experience relapse and do not survive beyond five years [Cytogenetic and molecular mutational features play an important role in AML risk stratification , 4. CompHere, we report a study that investigated the prevalence, features, and clinical correlation of cytogenetic and mutational characteristics of CK-AML in a cohort of 284 children with AML. Our study showed CK was associated with decreased survival in childhood AML and its impact on outcome correlated with the number of chromosomal aberrations. These results would aid in informing risk stratification of childhood AML to guide risk-adapted therapy.A total of 284 patients (≤ 18 years old) with de novo AML were enrolled in the study between 2007 and 2018 at Children’s Hospital of Chongqing Medical University in China. The diagnoses were based on histological, cytogenetics, and immunophenotyping analyses of bone marrow. The patients were treated with daunorubicin/cytarabine/etoposide (DAE)-based regimen following the protocols of the Pediatric Hematology Group of Chinese Medical Association . The stuRUNX1/RUNX1T1/t, CEBP/MYH11/inv(16)(p13.1q22) /t ; PML/RARA/t; BCR/ABL1/t, and KMT2A(MLL)/11q23 rearrangement . A complex karyotype was defined as three or more chromosomal aberrations in the absence of the WHO recurrent AML genetic aberrations of t, inv(16)(p13.1q22) /t ; t, t, KMT2A(MLL)/11q23 rearrangement, and t [G-banded karyotyping and fluorescence in situ hybridization (FISH) studies were performed according to the standard procedures . A compl4;q11.2) . An unba4;q11.2) . Down sy4;q11.2) .NPM1,and/or biallelic mutated CEBPA; high-risk factors were -7, -5/5q-, t, t, inv(3)(q21q26.2)/t, KMT2A/11q23 rearrangement except t, and FLT3-ITD; and intermediate-risk features were those without the high or low-risk features [Risk classification was following the modified US Children Oncology Group (COG) AML risk stratification scheme: low risk features included t, inv(16)(p13.1q22) /t ; t, mutated features , 23, 26.FLT3 (exons 14–15), NPM1 (exon 12), WT1 (exons 7 and 9), NRAS (exons 1–2), KRAS (exons 1–2), IDH1 (exon 4), IDH2 (exon 4), KIT , CEBPA (exon 1), CCDN1 (exon 5), ASXL2 (exons 11–12), DHX15 (exon 3), GATA2 (exons 4–6), and DNMT3A (exon 2) [Total RNA was extracted from bone marrow samples using the Tiangen RNAprep Pure Blood Kit , and used as the template for cDNA synthesis with the Reverse Transcription System . DNA fragments covering the mutational hotspots were polymerase chain reaction (PCR) amplified from cDNA following the conditions previous described –32. The (exon 2) –32.2), Fisher’s exact, or Mann–Whitney U test, as appropriate. Complete remission (CR) was defined as bone marrow with less than 5% blasts and evidence of regeneration of normal hematopoietic cells. Overall survival (OS) was calculated from the date of diagnosis to death or last contact. Event-free survival (EFS) was the time between diagnosis and occurrence of the first event . OS and EFS were estimated using the Kaplan–Meier analysis, and the differences were compared using the log-rank test. A p value of ≤ 0.05 (two-sided) was considered statistical significance. The analyses were performed with SPSS software package v17.0 .Patient characteristics were compared using chi-square (χp = 0.025). Sixteen CK cases harbored three to four abnormalities and the others had five or more aberrations. Multiple cytogenetic clones defined as two or more clones were observed in 17 (50%) patients with CK-AML.Of 284 patients in the cohort, 225 (79.2%) cases showed clonal cytogenetic aberrations. One hundred forty-three were classified as low-risk, 109 intermediate-risk, and 32 high-risk. Thirty-four (12.0%) patients met the criteria for CK    followed by CEBPA, FLT3/ITD, and IDH1 genes (6.0% each), and none was observed in NPM1, KIT, CCND1, IDH2, ASXL2, DHX15, and DNMT3A genes (Table p = 0.08) while there were no differences in other clinical and molecular features between the two groups  and had a higher percentage of blasts in marrow  compared to those with two or more clones, but no difference in gene mutational frequencies (Table CEBPA and NKRS)    but no difference in EFS  compared with those with atypical CK-AML. Patients with ≥ 5 chromosome aberrations had an inferior OS and EFS  than ones with ≤ 4 aberrations, indicating a positive correlation between the number of chromosomal aberrations and worsen survivals. No differences in CR and relapse rates, as well as survivals, were observed between CK with a single and multiple clones  . Compared to those with intermediate-risk AML, patients with CK-AML seemed less likely to reach complete remission  [WT1 gene mutation with outcome information available, two relapsed and died at 8 and 26 months, respectively, after diagnosis. These were similar to the observations reported by others that mutant WT1 gene is associated with decreased survivals and high relapse [TP53 gene aberrations have been reported in 40%-50% of adult patients with CK-AML [TP53 mutational analysis was not performed in the present study, and we only observed one CK-AML case (2.9%) with a 17p13.1 deletion by cytogenetic analysis. Taken together, our results show a difference in cytogenetic and mutational profiles between childhood and adult CK-AML, which is in accordance with findings in other AML subtypes [Thus far, there is limited information on mutational profile of CK-AML and no mutational profiling of childhood CK-AML has been reported in the literature . The ress. 1.5%) . Of thre relapse , 45. Finh CK-AML , 46. TP5subtypes .Differences in results between our study and others are likely attributed to variation in the composition of study cohorts including the number of patients, age, treatment modalities, criteria for complex karyotype (≥ 3 vs. ≥ 5 aberrations), geographic locations, ethnic groups, methods used in mutation analysis, and the number of genes examined. Our results need to be validated by future studies of large cohorts of children with CK-AML.To the best of our knowledge, no such studies have been reported in the literature and ours is the first in the Chinese population. Our results demonstrate for the first time that among Chinese children with CK-AML, atypical CK was more frequent than typical CK, mutational incidences were low and concomitant mutants were uncommon. CK-AML had reduced EFS and OS compared with intermediate-risk AML, indicating CK as an adverse risk marker for childhood AML. Typical CK-AML tended to correlate with decreased OS compared to atypical CK-AML. Moreover, CK-AML with five or more cytogenetic aberrations was associated with inferior survivals than CK with four or fewer abnormalities, suggesting that the number of cytogenetic abnormalities in CK may influence outcome. Results from our study would inform refinement of risk stratification for childhood AML to improve outcomes.Additional file 1. List of complex karyotypesAdditional file 2. Comparison of clinical and molecular features of childhood CK-AML with typical and atypical CKAdditional file 3. Comparison of clinical and molecular features of childhood CK-AML with ≤4 or ≥5 cytogenetic aberrations


We found that store-operated Ca2+ entry (SOCE) formed by Ca2+-selective channel ORAI1 and Ca2+ sensors STIM1/STIM2 contributes to Piezo1-induced Ca2+ influx in eMSCs. Particularly, the Yoda1-induced increase in intracellular Ca2+ ([Ca2+]i) is partially abolished by 2-APB, a well-known inhibitor of SOCE. Flow cytometry analysis and wound healing assay showed that long-term activation of Piezo1 or SOCE does not have a cytotoxic effect on eMSCs but suppresses their migratory capacity and the rate of cell proliferation. We propose that the Piezo1 and SOCE are both important determinants in [Ca2+]i regulation, which critically affects the migratory activity of eMSCs and, therefore, could influence the regenerative potential of these cells.Endometrial mesenchymal stem cells (eMSCs) are a specific class of stromal cells which have the capability to migrate, develop and differentiate into different types of cells such as adipocytes, osteocytes or chondrocytes. It is this unique plasticity that makes the eMSCs significant for cellular therapy and regenerative medicine. Stem cells choose their way of development by analyzing the extracellular and intracellular signals generated by a mechanical force from the microenvironment. Mechanosensitive channels are part of the cellular toolkit that feels the mechanical environment and can transduce mechanical stimuli to intracellular signaling pathways. Here, we identify previously recorded, mechanosensitive (MS), stretch-activated channels as Piezo1 proteins in the plasma membrane of eMSCs. Piezo1 activity triggered by the channel agonist Yoda1 elicits influx of Ca Mesenchymal stem cells derived from a wide range of adult tissues, such as bone marrow, umbilical cord, peripheral blood and adipose tissue, are widely applied to treat various diseases. In the past decades, new sources of adult stem cells, including the placenta , fallopi2+ ions, which are known to be crucial, secondary messengers in living cells. There is substantial evidence connecting the handling of intracellular calcium concentration ([Ca2+]i) to the normal physiology and pathophysiology of endometrial stromal cells. It is established that different Ca2+-permeable ion channels are involved in decidualization and play a key role in embryonic functions i is tightly connected with uterine function [2+]i level regulation in endometrial cells. Given the significant role of the temporal and spatial Ca2+ regulation in a variety of main cellular functions, including cell motility, we also examined the role of Piezo1 in endometrial cell migration.The participation of Cafunction ,26,27,282+ influx and, thereby, affect the [Ca2+]i and Ca2+-dependent signaling processes. By consistently using PCR analysis and immunofluorescence, we first confirmed the expression of Piezo1 in eMSCs  after the addition of Yoda1 to the external solution. Then, several minutes later, a second increase in [Ca2+]i (delayed component), which lasted the length of the [Ca2+]i registration, was observed  via the plasma membrane , a specific, irreversible inhibitor of endoplasmic reticulum (ER) Ca2+-ATPases in the same ion conditions resulted in TG-induced Ca2+ release, as was previously documented elsewhere i. Subsequent application of TG caused a rapid [Ca2+]i growth followed by a rapid decline  one, suggesting the SOCE contribution in Piezo1-mediated Ca2+ increase.Therefore, to eliminate the potential contribution of SOCE in Piezo-induced Ca2+ signaling is important for effective cell migration. However, how Ca2+ coordinates the necessary factors for effective cell moving, key signaling pathways and signaling molecules is still elusive. Here, we investigate the likelihood of participation of Piezo1- or SOCE-induced Ca2+ currents in eMSC migration.It is well known that MSCs possess a homing capacity, can move into damaged areas and help in tissue regeneration ,39,40,41The wound healing assay, together with time-lapse imaging, was used to address this challenge. To avoid some possible, uncontrolled effects caused by cell damage, the wound in the eMSC culture was created by the removal of a silicone insert, as described in Materials and Methods. Wound healing is a standard behavior of epithelial and endothelial cells, as well as mesenchymal cells, that is firstly activated to increase their motility in order to settle the injured area and then, together with their ability to differentiate and to secrete various biologically active factors and molecules , they al2+ currents on a millisecond time scale. It appears that these evidently specific force transducers exist throughout the numerous cells. Although Piezo channels discovery happened relatively recently, extensive evidence has already been found for their significance in many aspects of cellular life.It is well known that all eukaryotic cells are mechanosensitive since they are subjected to various environmental mechanical forces, such as gravity, tension, compression and shear. These mechanical forces impact various cellular functions down to the fate definition of cells. The mechanosensitive Piezo channels are capable of sensing mechanical force and transform force and pass signals into cells in the form of Ca2+ entry in eMSCs. Importantly, the Yoda1-induced Ca2+ entry has two distinct components that are separated in time. The kinetics of Yoda1-induced Ca2+ response are similar to those induced by TG in numerous cells, particularly: (i) TG-mediated [Ca2+]i growth caused by store depletion followed by (ii) store-operated Ca2+ entry. However, our experiments demonstrated that the application of Yoda1 in a Ca2+-free solution has no effect on [Ca2+]i, whereas the following addition of TG results in the increase of [Ca2+]i.Here, we report that the mechanically gated ion channel Piezo1 is expressed in human endometrial stem cells. We also show that the small-molecule modulator of Piezo1 channels, Yoda1, induces large Ca2+ entry were detected in Ca2+ measurements carried out on eMSCs. We also identified that store-operated Ca2+ entry in eMSCs could be formed by ORAI1/STIM1 or ORAI1/STIM2 proteins. In our experiments, a specific blocker of SOCE, 2-APB, suppressed the TG-induced SOCE. Moreover, 2-APB also blocked the second (delayed) component of Yoda1-induced Ca2+ increase, suggesting the SOCE is participating in mechanosensitive Ca2+ signaling in cells. These findings correlate with the data which show that store-operated Ca2+ entry can participate in Ca2+ influx induced by the other agonists, for example, ATP -5-pyrazin-2-yl-1,3,4-thiadiazole (Yoda1) was purchased from Tocris . 2-aminoethyl diphenylborinate (2-APB) and thapsigargin (TG) were purchased from Sigma-Aldrich .The endometrial mesenchymal stem cell (eMSC) line isolated from the desquamated endometrium of the menstrual blood of a healthy donor  was a g2, 1 unit Hot-Taq polymerase and 1 × Hot-Taq polymerase buffer . In negative control experiments, MMLV reverse transcriptase was omitted. PCR reaction products were subjected to electrophoresis on a 6% polyacrylamide gel and visualized by UV fluorescence after ethidium bromide staining.Total RNA was isolated using the RNeasy Mini Kit . The PCR primers were designed using the GeneRunner v5.0.59 software. The primer sequences are represented in ORAI1  or Abs against STIM1  or STIM2  at 4 °C overnight. Goat anti-rabbit (GAR)-Cy3 Abs  were used for secondary detection. Cell nuclei were counterstained with DAPI  and then the samples were mounted using Vectashield mounting medium . As a control, staining of the cells only with fluorescent secondary Abs  was used. Images were obtained using the Olympus FV3000 laser scanning confocal inverted microscope  with 40×/1.3 NA oil immersion objective and a specific combination of laser/detectors.For immunofluorescent staining, the cells, preliminary plated on glass coverslips, 2–3 days before the experiments, were fixed with 3.7% paraformaldehyde in phosphate-buffered saline  and permeabilized with 0.25% Tween-20 . Non-specific binding of the antibodies (Abs) was blocked by incubation with 10% goat serum for 1 h at RT. Then cells were incubated with goat anti-rabbit polyclonal Abs against Piezo1  or rabbit polyclonal Abs against 2, 1 MgCl2 and 10 HEPES/TrisOH. Potassium bath solution containing 145 KCl, 2 CaCl2, 1 MgCl2 and 10 HEPES/TrisOH was used to nullify the resting membrane potential of the cells. The pH of all solutions was maintained at 7.3. All experiments were performed at RT. The single-channel recordings were processed and analyzed in Clampfit software (part of Axon PClamp 10.7 Software Suite). Single-channel conductance values were defined by the slope of the current–voltage relationship (I–V) after linear approximation. Averaged conductance values are given as the mean ± SEM (n—number of experiments).Single currents were recorded using cell-attached configuration, as described previously . Briefly2+ measurements. On the day of the experiment, cells were washed with a serum-free medium and loaded for 35 min with a 4 μM Fura-2AM probe  in the dark at RT. Then, cells were washed, and the cover slides were transferred into a perfusion chamber. Cell imaging was obtained using an AxioObserverZ1 inverted microscope  with a Carl Zeiss Fluar 40×/1.3 Ph3 NA oil objective. Fura-2AM fluorescence was excited every 5 s sequentially by the light of 340 nm or 380 nm from an illuminator with a Lambda DG-4 high-speed wavelength switcher . Filter set 21 HE  was used . “Physiology” plugin of AxioVision 4.8.2 software  was used for data acquisition and analysis. Calcium imaging recordings were subjected to the subtraction of the background fluorescence. Fura-2AM 340/380 nm ratio values are given as mean ± SD (n—number of the cells).eMSCs were seeded on cover slides 2–3 days before Ca2). The images of the experimental wounds were acquired automatically  using the “Multidimensional Acquisition” plugin of Carl Zeiss Axiovision 4.8.2 Software . The sizes of experimental wounds were calculated manually using the “Measure Area” tool in ImageJ software , and the wound sizes were normalized to the size of the initial wound (at 0 h starting measurement). Wound sizes are shown as mean ± SEM (n—number of independent experiments). Data were compared using a two-sample Student’s t-test ; p < 0.05 was considered as significant.For the wound healing assay, trypsinized eMSCs were seeded into Ibidi Culture-Inserts 2 Well  pre-installed in 24-well culture plates to form the experimental wounds (gaps of the standardized sizes). The cells were cultured for a further 24 h to allow cell spreading, then, the inserts were removed, and the plates were transferred to the stage of a Carl Zeiss AxioObserver Z1  microscope equipped with the set-up for long-term live-cell imaging under stable culture conditions  and are presented as mean ± SD.Detached with trypsin/EDTA solution, eMSCs were suspended in growth medium and utilized for viability and cell cycle analysis as described previously . Brieflyp < 0.05 was considered as significant.Statistical analysis was performed using GraphPad Prism 6.0 software . All data were tested for normal distribution using Shapiro–Wilk’s test and homogeneity of variances (Levene’s test). The particular, statistical criteria used for means comparison are described in the relevant sections of Materials and Methods;


However, whether SERCA2a inactivity induces cytosolic Ca2+ imbalance in mitochondrial homeostasis is far from clear. Mitofusin2 (Mfn2) is well known as an important protein involved in endoplasmic reticulum (ER)/mitochondrial Ca2+ tethering and the regulation of mitochondrial quality. Therefore, the aim of our study was to elucidate the specific mechanism of SERCA2a-mediated Ca2+ overload in the mitochondria via Mfn2 tethering and the survival rate of the heart under conditions of cardiac microvascular ischemic injury. In vitro, CMECs extracted from mice were subjected to 6 h of hypoxic injury to mimic ischemic heart injury. C57-WT and Mfn2KO mice were subjected to a 1 h ischemia procedure via ligation of the left anterior descending branch to establish an in vivo cardiac ischemic injury model. TTC staining, immunohistochemistry and echocardiography were used to assess the myocardial infarct size, microvascular damage, and heart function. In vitro, ischemic injury induced irreversible oxidative modification of SERCA2a, including sulfonylation at cysteine 674 and nitration at tyrosine 294/295, and inactivation of SERCA2a, which initiated calcium overload. In addition, ischemic injury-triggered [Ca2+]c overload and subsequent [Ca2+]m overload led to mPTP opening and ΔΨm dissipation compared with the control. Furthermore, ablation of Mfn2 alleviated SERCA2a-induced mitochondrial calcium overload and subsequent mito-apoptosis in the context of CMEC hypoxic injury. In vivo, compared with that in wild-type mice, the myocardial infarct size in Mfn2KO mice was significantly decreased. In addition, the findings revealed that Mfn2KO mice had better heart contractile function, decreased myocardial infarction indicators, and improved mitochondrial morphology. Taken together, the results of our study suggested that SERCA2a-dependent [Ca2+]c overload led to mitochondrial dysfunction and activation of Mfn2-mediated [Ca2+]m overload. Overexpression of SERCA2a or ablation of Mfn2 expression mitigated mitochondrial morphological and functional damage by modifying the SERCA2a/Ca2+-Mfn2 pathway. Overall, these pathways are promising therapeutic targets for acute cardiac microvascular ischemic injury.Our previous research has shown that type-2a Sarco/endoplasmic reticulum Ca Acute myocardial infarction (AMI) refers to a clinical or pathological event with evidence of myocardial injury due to acute myocardial ischemia . AlthougTan et al., 2020b2+ back to the ER . All mice were bred on a C57BL/6 background for at least 3 generations. Subsequently, 8-week-old wild-type (WT) and Mfn2KO mice were used to establish a heart ischemic injury model according to our previous studies with minor modifications . The Mfn2 loxp/loxp mice with cardiomyocyte-specific deletion of the mfn2 exon were generated based on the Cre-Lox strategy . Brieflyications . For theications .CMECs were isolated from C57BL/6 background WT mice (6 months old) using the enzyme dissociation method as previously reported with minor modifications . In brie2 incubator at 37°C for 6–8 h. The subsequent experiment was conducted after the cells had been cultured for 24–48 h. A synthetic Mfn2 gene fragment was introduced by Hua Yueyang Biotechnology Co., Ltd. . Two sets of Mfn2-siRNA were designed and had the following sequences: 5′-GGACCCAGTTACTACAGAAGA-3′ and 5′-GCTCCTGGCTCAAGACTATAA-3′. The control gene was UUCUCCGAACGUGUCACGUTT (primer sequences: 5′–3′).The SERCA2a gene adenoviral vector was used to overexpress SERCA2a in CMECs. A non-cell-type-specific recombinant adenovirus vector carrying the SERCA2a gene  and an empty recombinant adenovirus vector  were constructed using the vector gene technique. Synthetic SERCA2a gene adenovirus vectors were injected by Hua Yueyang Biotechnology Co., Ltd. . Next, we established siRNA-Mfn2 in CMECs . Twenty-in vitro as previously described  containing 5% fetal bovine serum  at 37°C to perform further experiments immediately  and immunofluorescence using a Leica confocal microscope . Cytoplasmic ROS was measured by DCFH-DA (Sigma) (t = 0), which was normalized by the control group. The TUNEL assay was used according to the manufacturer’s instructions to detect cell death after hypoxic injury. For quantification, the numbers of TUNEL-positive cells were calculated in at least 10 random separate fields to obtain the average percentage in different groups  or Rhod-2  for 30 min. After washing with PBS, the intensities of cytoplasmic Ca (Sigma) . ImageJ t groups .Briefly, isolated hearts were homogenized in buffer containing 10 mM imidazole (pH 7.0), 300 mM sucrose, 1 mM EDTA, 1 mM or 10 mM DTT and 0.3 mM phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 8,000 g for 20 min. The supernatant was removed, adjusted to a final concentration of 600 mM and centrifuged at 100,000 g for 1 h. The pellet was resuspended in homogenization buffer with 600 mM NaCl and centrifuged again at 100,000 g for 1 h . The pelCMEC mitochondria were isolated as described previously . The mPTAfter treatment, the samples were precleaned with Protein A/G (Santa Cruz Biotechnology) and incubated with antibodies as follows. Then, Protein A/G was added, and the samples were shaken at 4°C overnight. The precipitates were washed three times with PBS, and the proteins were denatured for Western blot analysis. The primary antibodies were anti-nitrotyrosine (NT)  and anti-SERCA2a  . The metSERCA2a and Mfn2 expression was assessed by immunofluorescence staining. Briefly, cells were collected and fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with 10% goat serum albumin  for 1 h at room temperature . SubsequEchocardiography was immediately performed in all mice after the heart ischemia model was established. Left ventricular fraction shortening (LVFS), left ventricular ejection fraction (LVEF), left ventricular dilated dimension (LVDD) and left ventricular volume-systole (LVV) were measured in M-mode images using computer algorithms .2 for 24 h. After washing in 0.1 M sodium cacodylate buffer, tissues were postfixed in 1% OsO4 and 0.1 M cacodylate buffer overnight, dehydrated and embedded in Embed 812 resin . Heart samples were fixed in 5% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) with 0.05% CaCl12 resin . The secBriefly, tissues were homogenized and sonicated in lysis buffer. Next, the samples were centrifuged for approximately 10 min at 4°C to obtain the supernatants. Equivalent amounts of protein from different groups were separated by SDS-PAGE and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane . The mem2+, the cell-permeable Ca2+ chelator BAPTA (Sigma) was used before the injury model. To detect the effect of ROS levels on different groups of CMECs, NAC (Sigma) was used as the negative group. tBHQ (Sigma) was used as a pretreatment to inhibit the expression of SERCA2a  in different groups. As shown in 2+]c intensity, while this effect was blocked by SERCA2a overexpression or pretreatment with the antioxidant reagent NAC. To confirm the effect of elevated Ca2+ levels on cell survival, we performed a TUNEL assay, and the results showed that there were more cell deaths in the hypoxic injury group than in the control group , can be filled with excessive Ca2+, resulting in [Ca2+]m overload and serious side effects. Therefore, we hypothesized that in CMECs with hypoxic injury, [Ca2+]m overload would be followed by pathologically excessive [Ca2+]c levels. First, we used the [Ca2+]m probe Fluo-3AM and the [Ca2+]c probe Rhod2 to detect the change in Ca2+ concentration in different groups by flow cytometry . Furthermore, we used qPCR to test the efficiency of Mfn2 knockout in mice   . Moreove < 0.05) . As showc volume , while tKO mice was improved compared with that in WT ischemic injury mice, the Mfn2KO mice still showed obvious fragmentation and swelling of mitochondria similar to that in the WT mice. Ischemic injury mice displayed abnormal red blood cell (RBC) morphology (the control RBCs were parachute-shaped), which indicated severe damage to microvessel integrity and barrier function  were analyzed using ELISAs, which indicated that ischemic injury induced higher inflammatory reactions  . Howeverfunction . To assefunction . In the eactions . However2+ homeostasis. It is highly sensitive to the loss of cellular energy and redox status and changes in cytosolic Ca2+ concentration, which ultimately lead to the accumulation of unfolded proteins, a condition known as ER stress  is a critical molecule for maintaining a balanced concentration of Ca2+ during the cardiac cycle, since it controls the transport of cytoplasmic Ca2+ to the sarcoplasmic reticulum (SR). The activity of this pump has been widely investigated, emphasizing its central role in the control of Ca2+ homeostasis and consequently in the pathogenesis of different cell types , is filled with excess Ca2+ in some diseases, causing [Ca2+]m overload followed by severe side effects. Due to mitochondrial calcium overload, ATP depletion, oxidative stress, and disrupted mitochondrial matrix pH, a series of events leads to mPTP opening, which leads to mito-apoptosis via the release of cytochrome c to the cytoplasm and the activation of the intrinsic caspase pathway  as a molecular tether that links SR to mitochondria (2+ overload, subsequently attenuating mitochondrial oxidative stress and inhibiting mPTP opening [30].Mitochondria/ER tethering is required for Cachondria . There achondria . Moreovechondria , 2012a. chondria . Other fchondria . MiR-497chondria . Strong chondria . Adult Mchondria . Therefo2+]c imbalance is the upstream factor of Mfn2-mediated Ca2+ transport tethering. In our in vivo experiment, the results proved that either knockout of Mfn2 or overexpression of SERCA2a decreased heart infarction size, improved mitochondrial morphology, improved acute heart infarction indicators such as LDH, Troponin T, and CK-MB, restored myocardial contractile function, and maintained morphological structural integrity.Furthermore, there have been no reports about the relationship between Mfn2 and SERCA2a inactivity in microvascular acute myocardial injury. We hypothesized that SERCA2a-induced c overload caused mitochondrial dysfunction and activated Mfn2-mediated [Ca2+]m overload. Knockout of Mfn2, the downstream target of SERCA2a, also suppressed fatal Drp-1-dependent mito-fission and Parkin/PINK-dependent mitophagy, which finally reduced heart ischemic injury in microvascular endothelial cells to some extent. In addition to impaired mitochondria/SR tethering, we provided a new mechanism for the role of Mfn2 inhibition during acute heart ischemia injury, which can be a novel strategy for microvessel cardioprotection.In summary, we have shown that ablation of Mfn2 rendered the heart resistant to heart infarct injury and reduced cardiac microcirculatory damage. We proved that SERCA2a-dependent [CaThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by the Sixth Medical Center of Chinese PLA General Hospital.YZ performed all the experiments. FT analyzed all the data. Both authors approved this submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Amlodipine, a unique long-lasting calcium channel antagonist and antihypertensive drug, has weak fluorescence in aqueous solutions. In the current paper, we show that direct visualization of amlodipine in live cells is possible due to the enhanced emission in cellular environment. We examined the impact of pH, polarity and viscosity of the environment as well as protein binding on the spectral properties of amlodipine in vitro, and used quantum chemical calculations for assessing the mechanism of fluorescence quenching in aqueous solutions. The confocal fluorescence microscopy shows that the drug readily penetrates the plasma membrane and accumulates in the intracellular vesicles. Visible emission and photostability of amlodipine allow confocal time-lapse imaging and the drug uptake monitoring. Theblockers . Recent blockers ,7. Its pthe cell . The lonmembrane . AML hasn plasma .0) and the lowest excited singlet state (S1). This is achieved by the rotation of 2-chlorophenyl ring followed by the DHP fragment deformation. Suppression of such large amplitude motion in AML, for example by a viscous medium, leads to the fluorescence enhancement. When bound to a protein, the motion of the ligand is severely limited [Our calculations show that the fluorescence loss in aqueous solutions is caused by the nonradiative relaxation and proceeds through the conical intersection between the ground state  increases (Φ) and the lifetime (τ) of AML fluorescence were significantly higher in the presence of BSA or HSA than for a free drug ree drug . The sell of AML . To studglycerol . The batglycerol .η); their dielectric constants 32.6 and 42.5 respectively  and non-radiative (nrk) decay constants illustrate this effect with 0k remaining almost constant and decreasing nrk with increasing viscosity of the medium  the calculated molecular structure of AML is similar to the one obtained by single crystal X-ray analysis [1, from which it emits a photon and the fluorescence occurs. The molecular geometry of AML in S1 does not change significantly from the one in the S0 -AML in the 0–750 µM protein (BSA or HSA) solutions and observed the fluorescence intensity changes at the emission maximum (dK) were in a good agreement with previous studies where the tryptophan (Trp) fluorescence quenching was used for the binding constant determinations [λem (Trp) = 350 nm helps to minimize autofluorescence.The enhancement of AML fluorescence upon binding to BSA or HSA allowed us to determine the AML-protein binding affinities by fluorescence titration experiments. For that, we recorded the fluorescence spectra of 5 µM AML or -(−)-Bay K8644.12. (S)-(−)-Bay K8644 had no effect on cell staining by AML , ER-Tracker® Red (endoplasmic reticulum); and genetic markers: Golgi-emGFP, Rab7a-emGFP (late endosomes), Rab5a-emGFP (early endosomes) and LAMP1-emGFP (lysosomes). We treated living A7r5 cells with the respective marker and AML, and then visualized using a confocal Leica SP8 microscope. After 1 h incubation, the late endosomal and lysosomal labelling showed the co-occurrence of the cyan (AML) and green channel (emGFP) emissions with calculated Pearson’s co-localization coefficients (R) above threshold: R ~0.4 -(–)-amlodipine ((S)-AML) besylate (98 +%) was purchased from Carbosynth . (S)-(−)-Bay K8644 was purchased from Tocris Bioscience . BSA (98 +%), HSA (Fraction V) and Phosphate Buffered Saline tablets for 0.01 M phosphate buffer (PBS) were purchased from Merck, Darmstadt, Germany. All reagents were used without further purification. To prepare 20 mL of 10% BSA/HSA solutions in phosphate buffered saline , 2 g of protein was dissolved in a 20 mL volumetric flask with the previously prepared PBS. The protein solution was kept in a refrigerator until use.Racemic amlodipine besylate (97+%) was purchased from Alfa Aesar .  at room temperature (25 °C). Fluorescence emission of AML was recorded from 390 nm to 600 nm with excitation at 360 nm. The emission bandwidth was set to 10 nm, excitation bandwidth—7.5 or 10 nm, gain—100, number of flashes—20. The absorption spectra were recorded: from 300 to 430 nm. The background absorption of a glass bottom plate was measured in wells containing only buffer and subtracted from the spectra of the samples. The spectra were averaged from three individual experiments. The absorption spectra of AML in glycerol/methanol mixtures were recorded on a Varian Cary 4000 UV-Vis spectrophotometer in quartz cuvettes (3 mL) with a 1 cm path length. Fluorescence spectra of 5 µM AML in PBS solutions of different pH (3–9) were recorded on a Varian Cary Eclipse fluorescence spectrometer.Φ) were obtained on a Quantaurus-QY absolute PL quantum yield spectrometer  at ambient temperature (25 °C), excitation wavelength 370 nm; all measurements were performed in triplicates. Fluorescence lifetimes (τ) were measured with a Quantaurus-Tau fluorescence lifetime spectrometer . All measurements were performed in air-saturated solvents at ambient temperature. Radiative fluorescence lifetime (radτ) was calculated by the formula: radτ = τ/Φ.Absolute fluorescence quantum yields . All transient spectra were recorded using 10 × 10 mm quartz cells with a capacity of 4 mL and were bubbled for 30 min with argon before acquisition. All the experiments were conducted at room temperature.The molecular geometry of the compound and its spectra were calculated using the FireFly program package  partiall−1 and f is its oscillator strength. The rate of achieving the conical intersection via the barrier was calculated by the Arrhenius formula: −1 and A is the barrier height.The radiative rate constant was calculated by the formula: v/v% DMSO). The obtained solutions were incubated for 1 h at room temperature to ensure ligand binding to the proteins. Emission spectra of BSA and HSA solutions, as well as the spectra of BSA/HSA solutions containing AML or (S)-AML (5 µM), were measured on a multiwell plate reader as given in “General materials and methods” section; the emission wavelength step size was 1–2 nm. The fluorescence intensities at 450 nm were measured for the calculation of the AML fluorescence increase upon binding to BSA/HSA. The fluorescence increase of compounds in BSA or HSA in relative fluorescence units (RFU) was plotted vs. proteins concentrations. Binding constants were calculated using the software package GraphPad Prism version 8.3.1 . Y = (Bmax × X/(dK + X)) + off was used, where Bmax is the maximum specific binding.The tested compounds were dissolved in the PBS containing different concentrations of BSA or HSA  were cultured in DMEM/F-12  supplemented with GlutaMAX-1 (Thermo Fisher Scientific), 10% fetal bovine serum (FBS)  and 0.9% Penicillin/Streptomycin  in a humidified 5% CO2 incubator at 37 °C. In addition, we used a reduced antibiotic pressure for the cultivation of the cells; 100 µg/mL hygromycin B , 15 µg/mL Blasticidin , 0.4 µg/mL puromycin . To induce the expression of CaV1.2, 0.1 µg/mL doxycycline  was added 24 h before experiments.HEK-293 Ca2 atmosphere at 37 °C. Cells were split every 3–5 days or at confluence.A7r5 cells  were cultured in DMEM Medium supplemented with 10% FBS, 0.9% penicillin/streptomycin in a humidified 5% COHL-1 cells  were cultivated in Claycomb’s Medium (Sigma), supplemented with Glutamax, 10% fetal bovine serum, 0.9% penicillin/streptomycin and 0.1 mM noradrenaline (Sigma) on Fibronectin/Gelatine (Sigma) coated culture bottles or plates.2+ assay, HEK-293 CaV1.2 cells  or HL-1 cells  were cultivated for 24 h in black-walled, clear-bottom 96-well microplates  covered with fibronectin . Cells were grown in a humidified incubator at 37 °C and 5% CO2.For fluorescent plate reader Ca2+ concentration were measured on a multiwell plate reader TECAN Spark 20M using FLIPR 6 Calcium Assay Kit from Molecular Devices  at room temperature (25 °C). The 1000× stock solutions of AML were prepared in DMSO. Briefly, the culture medium was removed and the 100 µL loading buffer was added to the cells together with 100 µL AML solutions of different concentrations in Hanks’ balanced salt solution  containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Then the cells were incubated for 2 h at 37 °C. The cells were washed 2 times with 200 µL HBSS containing AML or (S)-AML (1 nM–5 µM) and 20 mM HEPES. The time courses were recorded in each well. Fluorescence was evoked by 485 nm excitation wavelength and collected in a bottom-read mode at 525 nm. Data was recorded every 2 s . To initiate Ca2+ influx, 50 µL of 400 mM KCl solution in HBSS was injected in each well (speed 100 µL/s) at 10 s time point. To get a fluorescence maximum, 15 of 30 µM Ionomycin  solution in HBSS was injected in each well (speed 100 µL/s) at 50 s time point. The ratios between the KCl induced peak minus basal fluorescence, ΔF(KCl), were taken and expressed as a quotient of the maximal increase induced by ionomycin, ΔF (Ionomycin). The dose response curves of AML were calculated using the software package GraphPad Prism version 8.3.1. All data are presented as mean ± SD in 4 independent experiments. Y = Bottom + (Top-Bottom)/(1 + 10^((LogIC50 − X) × HillSlope)) was used where HillSlope describes the steepness of the curve, Top and Bottom are plateaus in the units of the Y axis.Changes in CaV1.2) cells per well. For the confocal imaging cells were seeded in CELLview™ Slides ; 8000 cells per well or in 35 mm glass bottom dish ; well 14 mm, glass thickness #1.5, 30,000 cells per well. The induction of HEK-293 CaV1.2 cells was done by 24 h incubation in doxycycline solution (0.1 µg/mL). For cellular imaging, cells were incubated at 37 °C with 0.1–2 µM AML prepared in Hanks’ balanced salt solution (HBSS) supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, from a 10 mM stock solution prepared in DMSO. For co-staining experiments cell were labeled with CellLight® Golgi-emGFP , MitoTracker® Orange, ER-Tracker® Red, CellLight® Early Endosomes-emGFP (fusion construct of Rab5a and emGFP), CellLight® Late Endosomes-emGFP (fusion construct of Rab7a and emGFP), BacMam 2.0  according to the manufactures instructions. 5 µM SynaptoRed C2®  was used for membrane staining. For the nucleus staining, we used 1 µM TMR-Hoechst and 100 nM 610CP-5-COOH-Hoechst probe [For the wide-field microscopy experiments cells were seeded in µ-24 well plates  or glass bottom 12-well plates  at 25,000 (A7r5 and HL-1) or 12,500  were excited with a 488 nm laser (5% intensity) and detected with a regular PMT in the 510–560 nm range. ER-Tracker® Red was excited with a 561 nm laser and detected with the Leica HyD detector set within the spectral range of 600–650 nm. SynaptoRed C2 (FM 4-64) was exited with a 405  or 561 nm laser and detected with the Leica HyD detector set within the spectral range of 590–650 nm. 610CP-5-COOH-Hoechst probe was excited with 610 nm laser and detected with a regular PMT in the 635–650 nm range. For wide-field imaging we used Lionheart FX automated microscope (Biotek) equipped with dry air objectives 4×, 20× and 40× and sample injection system. 16 fields of view in 3 focusing planes, spanning 6 μm in thickness, were acquired per well. Image stitching and focus stacking was performed using in-built Gene 5 software (Biotek). The final images encompassed 1380 × 950 μm field of view and contained at least 220 and up to 2000 cells.Confocal imaging was performed on a Leica SP8  inverted confocal microscope equipped with an HC PL APO CS2 63x/1.40 Oil objective. All live-cell images shown in the main text were acquired using a 700 Hz bidirectional scanner, and a 92.26 × 92.26-μm field of view (1024 × 1024 pixels and 90 nm pixels), pinhole of 95.6 µm diameter (1 AU), pixel dwell time—325 ns. AML was excited with 405 nm laser (3% intensity) and detected with a regular PMT in the 420–470 nm range. CellLighthttp://svi.nl, accessed on 19 February 2021). All acquired or reconstructed images were processed and visualized using Huygens Essential 20.10 or ImageJ 1.53c software [The images in software . Co-loca


KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML.Epigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed-lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of the KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing downregulation of a  Inhibitors that target epigenetic modulators to rectify epigenomic abnormalities represent valid therapeutic strategies. Further understanding of how epigenetic dysregulation in AML contributes to leukemogenesis may uncover tractable therapeutic targets and biomarkers for AML patient treatment and/or prognostic evaluation.Epigenetic regulators are frequently mutated in acute myeloid leukemia (AML), leading to epigenomic alterations4. PAFc mediated recruitment of the H3K9 methyltransferases, such as SETDB1 antagonizes Mixed-Lineage Leukemia (MLL) signaling, and inhibition of SETB1 promotes AML cell proliferation6.Polymerase-associated factor 1 (PAF1), a core component of the PAF complex (PAFc) is essential in AML7 positing an oncogenic role for H3K9me3 demethylases in AML. Cheung et al. identified an H3K9me3 demethylase, KDM4C as a cofactor of the PRMT1 complex in MLL rearranged (MLLr) and MOZ-TIF2 AML8. Simultaneous knockout (KO) of all three members of the Kdm4 family (kdm4a/b/c) in mice attenuates MLL-AF9 AML9, indicating roles for the Kdm4 family in murine myeloid leukemia. However, the therapeutic benefit of targeting the KDM4 family in human AML is not well understood.Primary AML blasts from patients with poor prognosis feature global H3K9me3 hypomethylation10. KDM4A has different roles in normal tissue development compared to other members of the KDM4 family; it is amplified/overexpressed in various malignancies including AML  activity, which can stratify high-risk patients. These findings support an essential and unique role of KDM4A for AML cell self-renewal and survival.Our previous lentiviral knockdown (KD) screen targeting epigenetic regulators in 12 human AML cell lines representing several AML subgroups found that KMD4A KD leads to significant suppression of leukemia cell proliferationAML Fig.  and corrdev, and lentiviral constructs for KD experiments   supplemented with appropriate cytokines as described13. Murine MLL-AF9 AML cells were leukemic BM cells extracted from a mice cohort with MLL-AF9 AML established by Somervaille et al.14, and cultured in the conditional medium with mIL-3 (100 ng/ml). Cytokines purchased from PeproTech .Leukemia cell lines were from DMSZ  and grown in RPMI (10% FBS and 2 mM L-Glutamine) at 37 °C in 5% COCell viability was measured by cell count using trypan blue dye  /hemocytometer or resazurin  with the Envision Fluorescent Reader (Perkin Elmer). Apoptosis was assessed using Annexin-V/Dead Cell Apoptosis Kit  and cell cycle analysis with PI/RNase Staining solution  as per manufactures’ instructions. Data were acquired using an LSRII flow cytometer and an Aria III flow cytometer  and analyzed using FlowJo software .6 cells and lysed on ice. Protein Lysates were quantified using Pierce Coomassie Plus Bradford and ran in NuPAGE™ 4–12% Bis-Tris Protein Gels (Invitrogen) as per products’ instructions. Only one loading control is used when Western blot analyses with multiple antibodies have been done sequentially in the same membrane.Cell pellets were suspended in fresh lysis buffer  at 30 μl per 104 cells per condition were incubated on poly-L-lysine coated Hendley-Essex 12 well glass microscope slides for 1 h before being fixed in 4% formaldehyde in PBS. The cells were permeabilized in 0.5% Triton-X-100 PBS followed by 2 h of blocking in 5% BSA, 0.2% Triton-X-100 TBS. Primary antibody was applied overnight in a humidified chamber at 4 °C. Appropriate secondary antibody (1:500 dilution) was applied for further 1 h incubation at room temperature after removal of a primary antibody using PBS 0.1% Tween 20. Antifade mountant with DAPI reagent (Thermo Fisher #P36962) was used to seal each sample and images were captured on the Zeiss Axioimager M1 Epifluorescence and Brightfield Microscope. CellProfiler v2.2.0 image analysis software (CellProfiler) was used to quantify IF signals.6 × 10+ hematopoietic stem and progenitor cells (HSPC) and AML patient cells were performed by plating 10000 cells and 3000 cells, respectively, on methylcellulose . CFU-GM (Granulocyte/Macrophage), M(Macrophage), and E(Erythroid) colonies were enumerated 10 days after seeding.Colony-forming cell (CFC) assay for murine cells was performed by plating 1000 cells on methylcellulose . Colony Assay for human CD3410. Primary AML patient samples for xenograft transplantation were unfractioned primary blasts from our and Manchester biobank collections. Control or KDM4A KD human AML THP1 cells or primary AML patient blasts were FACS sorted 40 h following lentiviral infection and immediately transplanted into sub-lethally irradiated (450 cGy) NSG mice of 6–8-week-old, mixed-sex  via tail vein injection.Mice experiments were approved by the local animal ethics board and performed under a project license issued by the United Kingdom Home Office, in keeping with the Home Office Animal Scientific Procedures Act, 1986. Non-obese diabetic. Cg-Prkdc scid Il2rgtm1Wjl/SzJ (NSG) mice were purchased from Jackson Laboratories  for transplantation as previously described® green-based fluorescent system was used to quantify dsDNA using the Applied Biosystems 7900 HT Fast Real-Time PCR system. Each qPCR plate included technical triplicates of each specific target alongside two housekeeping genes (GAPDH and ß-Actin). Delta-Delta CT method was used for analysis of gene expression against control. RNA-seq and ChIP-seq reads were mapped to the hg19 human genome or mm9. Transcript abundances were calculated in transcripts per million (TPM) using Kallisto15. SICER was used for peak calling on default settings. Both data files are available in the Gene Expression Omnibus (GEO): GSE125376.RNA was extracted using QIAshredder™ columns and RNeasy Plus Microkit™ (Qiagen). RNA-seq libraries were produced using the TruSeq® stranded mRNA kit (Illumina) and sequenced on the Illumina NextSeq™ 500 platform. For ChIP-seq, DNA was purified using Diagenode’s iPure kitv2 and libraries made using the TruSeq ChIP Library Preparation Kit according to the manufacturer’s instructions. For QPCR, reverse transcription was carried out using Invitrogen SuperScript III First-Strand Synthesis kit. A SYBRKDM4A-9 signature score is calculated as shown below using the least absolute shrinkage and selection operator (LASSO) linear regression17.xi and ri denote the gene expression and coefficient of the ith gene  in the signature, respectively. Specifically, KDM4A-9 score = (TPM2 × 0.097292) + (CD82 × 0.038719) + (SLC29A2 × 0.167487) + (INF2 × 0.150025) + (STAR × −0.13354) + (ACP6 × 0.04633) + (IFI6 × 0.015753) + (MROH6 × 0.086738) + (GSDMD × 0.008289).The 17 were analyzed using the lumi 2.36.0 package. Intensity values were normalized by Robust spline normalization. The diagnostic capability of each gene signature to predict LSC activity across AML samples (GSE76008) was assessed by ROC (Receiver operating characteristic) curve analysis. The Youden index was used to identify the optimal cut-off value.LSC enrichment classification and raw expression data from AML samples (GSE76008)KDM4A-9 and LSC17 scores was performed prior to the linear summation of KDM4A-9 and LSC17 scores for each patient to generate a combined KDM4A-9/LSC17 score.Min-Max scaling of the LSC17 and KDM4A-9 signature genes, as well as KDM4A and PAF1 in AML patients, a filtered edge list (TOM ≥ 0.05)18 was constructed. An undirected network graph was generated using the graph.edgelist function from the igraph 1.2.5 package.To visualize the connections between the http://meme-suite.org/tools/meme-chip).Motif enrichment analysis of 400 bp length DNA sequences centered over the TSS of KDM4A or PAF1 bound (FDR ≤ 0.01) and DE  genes was performed using MEME-ChIP   Fig. . Consiston8 Fig. . CFC potred Fig. . KDM4A Ksis Fig.  and S1D sis Fig. . These rsis Fig.  and murisis Fig. . Importalls Fig.  or primalls Fig.  into reclls Fig. . Loss of KD Fig. . Togethekdm4a KD virus 20 23. These displayed significant inhibitions of cell proliferation in THP1 cells and primary AML patient blasts, inducing differentiation and apoptosis  genes were significantly deregulated following depletion of KDM4A (Log2 fold change (FC) ≥0.5 or ≤−0.5; adj. p ≤ 0.05; Fig. 24 and H3K27me325  and revealed significant enrichment of genes regulated by PAFc28 Fig. . This isipt Fig. C and D aipt Fig.  level insis Fig. E–G; S4B SEA and rsis Fig. . Togethe26, MLL-AF929, and KDM4A binding sites . Although the expression of HOXA9 itself was not affected by either KD, our data suggest KDM4A and PAF1 co-regulate their downstream targets in a parallel manner.Furthermore, genes with significant expression changes following KDM4A silencing were also enriched in direct PAF1 target genes26 Fig. , suggest26 Fig. . Furtherm32 Fig.  and pro-KDM4A expression is highly associated with PAF1 expression in patient datasets  achieves an optimal balance between specificity and sensitivity  downstream of the ore Fig. . High KDrts Fig. E–H indepSC+ Fig. , therefoity Fig. . Interesity Fig.  overcomiS downstrKDM4A-9 genes, whereupon H3K9me3 and H3K27me3 are enriched after KDM4A KD  ≥0.05)) with KDM4A as a highly connected node 37 which plays an important role in AML. Corroborating recent findings6, our data suggest KDM4A regulates H3K9me3 to direct leukemogenesis. H3K9me3 has emerged as a key player in repressing lineage-inappropriate genes, impeding the reprogramming of cell identity during development and cell fate determination38. It would be interesting to determine further the clinical diagnostic relevance of H3K9me3 in relation to KDM4A in AML patients.Supplemental MaterialSupplemental gene expression excel file


Humans have four genes that code for PMCA isoforms (PMCA1-4). A large diversity of PMCA isoforms is generated by alternative mRNA splicing at sites A and C. The different PMCA isoforms are expressed in a cell-type and developmental-specific manner and exhibit differential sensitivity to a great number of regulatory mechanisms. PMCA4 has two A splice variants, the forms “x” and “z”. While PMCA4x is ubiquitously expressed and relatively well-studied, PMCA4z is less characterized and its expression is restricted to some tissues such as the brain and heart muscle. PMCA4z lacks a stretch of 12 amino acids in the so-called A-M3 linker, a conformation-sensitive region of the molecule connecting the actuator domain (A) with the third transmembrane segment (M3). We expressed in yeast PMCA4 variants “x” and “z”, maintaining constant the most frequent splice variant “b” at the C-terminal end, and obtained purified preparations of both proteins. In the basal autoinhibited state, PMCA4zb showed a higher ATPase activity and a higher apparent Ca2+ affinity than PMCA4xb. Both isoforms were stimulated by calmodulin but PMCA4zb was more strongly activated by acidic lipids than PMCA4xb. The results indicate that a PMCA4 intrinsically more active and more responsive to acidic lipids is produced by the variant “z” of the splicing site A.The plasma membrane Ca By taking Ca2+ out of the cell, the PMCA modulates the resting cytosolic Ca2+ and is fundamental for shaping and ending the Ca2+ signals  are mostly responsible for removing Ca2+ from the cytosol, while the function of PMCA seems more suited for the control of Ca2+ dependent microdomain processes  with a calculated molecular mass of 135 kDa and an average sequence identity of about 80%   responsive region, in the segment that links domain A with the transmembrane helix M3 , L-α-phosphatidylcholine type XVI-E (Sigma) from fresh egg yolk, brain extract (BE) Type I Folch Fraction I from bovine brain containing approximately (w/w) 10% PI, 50% PS, and other lipids, phosphodiesterase 3′, 5′ cyclic nucleotide activator  from bovine brain, calmodulin-agarose, ATP , sodium dodecyl sulfate (SDS), and all other chemicals were obtained from Sigma.Yeast synthetic drop-out media supplement without leucine, yeast nitrogen base without amino acids, dextrose, polyoxyethylene 10 lauryl ether . The plates were allowed to grow for 72 h at 28°C.K616 cells    was gently stirred for 5 min with 5 μl of a sonicated mixture of 57 mg/ml C12E10 with 29 mg/ml PC or the indicated amount of BE. The suspension was thoroughly mixed and preincubated for at least 10 min on ice before being added to the ATPase reaction. The PMCA was incubated for 3 min at 37°C with the reaction medium before the initiation of the reaction by adding 3 mM [γ-32P]ATP. The reaction was finished by acid denaturation after 30 min. During this period, the amount of Pi released from ATP increased linearly with time.The CaProteins were electrophoresed on a 7.5% acrylamide gel according to Laemmli Laemmli,  and reveWestern blotting was performed as previously described . The same model based on the Hill equation was fitted to PMCA4xb and PMCA4zb and the statistical significance of the difference between datasets was assessed by the S. cerevisiae strain K616, the genes coding for Ca2+ ATPases PMR1, PMC1, and the regulatory subunit of calcineurin (CNB) have been inactivated , but this phenotype can be rescued by the expression of a high-affinity Ca2+ transport system. We have previously shown that the expression of PMCA4xb does not allow the growth of K616 cells in Ca2+ depleted media and successful complementation requires the expression of hyperactive forms of the protein, in which the autoinhibitory mechanism has been disrupted Activated versions of PMCA4xb can be produced either by introducing mutations in the A domain or by the removal of the C-terminal autoinhibitory segment, as in the case of the mutant CT120 lacking the C-terminal 120 amino acid residues  before measuring the Ca2+ signaling. Most cells express simultaneously a variety of PMCA variants, and for understanding the role of PMCA, it is necessary to advance the knowledge of each PMCA protein, their basal activity and how they respond to activators , which was indicated as the acidic lipid-binding site  and the other resulting in the release of the C-terminal autoinhibition (splice site C) are not independent but cooperate for regulating the activity of the PMCA.2+ signal in cells expressing the variant “z” in normal and pathological conditions.In this study, we found that PMCA4z a previously not characterized variant of PMCA originated by splicing at site A is more active and more sensitive to activation by acidic lipids than the more common form of PMCA4x. Future work will be needed to assess the relevance of this finding for the control of the CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.GRC, LRM, GDP, LR, and FTP performed experiments and analyzed data. GRC, FTP, LRM, and HPA designed research and analyzed data. HPA conceived the project and wrote the manuscript with comments from all the authors. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To clarify the protective effect of simvastatin on myocardial ischemia reperfusion injury (MIRI) and the underlying mechanism.  The MIRI model in rats was firstly constructed. Twenty-four male rats were randomly assigned into the sham group, ischemia-reperfusion (I/R) group, and simvastatin group, with 8 rats in each group. Contents of superoxide dismutase (SOD) and malondialdehyde (MDA), as well as serum levels of CK and inflammatory factors, in rats were determined by the enzyme-linked immunosorbent assay (ELISA). Lactate dehydrogenase (LDH) activity in the three groups was examined. Through flow cytometry and Cell Counting Kit-8 (CCK-8) assay, apoptosis and viability in each group were detected, respectively. Relative levels of HMGB1, Kruppel-like factor 2 (KLF2), eNOS, and thrombomodulin (TM) were finally determined.  Simvastatin treatment markedly enhanced SOD activity and reduced contents of MDA, LDH, and creatine kinase (CK) in MIRI rats. The increased apoptosis and decreased viability following MIRI were partially reversed by simvastatin treatment. Besides, MIRI resulted in the upregulation of inflammatory factors and chemokines. Their elevations were abolished by simvastatin. In MIRI rats, simvastatin upregulated KLF2 and p-eNOS.  Simvastatin protects inflammatory response at post-MIRI through upregulating KLF2, thus improving cardiac function. Myocardial ischemia reperfusion injury (MIRI) is a cardiovascular disease manifested as acute angina owing to stenosis of the coronary artery and myocardial blood supply disorder . MIRI ocStatins (HMG-CoA reductase inhibitors) are a class of cholesterol-lowering drugs used for preventing and treating cardiovascular diseases . In addiKruppel-like factor 2 (KLF2) is a transcription factor induced by laminar flow in vascular endothelial cells, which is a vital regulator in vascular endothelial functions –11. KLF2A total of 24 male Wistar rats (250-300 g) were habituated in a standard environment with free access to food and water. Animal procedures were conducted after 12 h food fasting. Rats were sacrificed at 6 and 24 h after reperfusion. This experiment was approved by the Ethic Committee of Zhengzhou University.24 male rats were randomly assigned into the sham group, I/R group, and simvastatin group, with 8 in each. Mechanical ventilation was conducted initially with 5 mL of tidal volume and 50 beats/min of respiratory rate. The rat heart was exposed through left thoracotomy at the third rib space. A 6.0 silk nontraumatic suture was passed through the epicardial layer around the main branch of the left coronary artery, with approximately 2 mm from its starting point. A plastic button with a diameter of about 5 mm was passed through the ligature and in contact with the heart. Both ends of the suture were passed through an exposed small vinyl tube. This method was convenient for generating MIRI in advanced preconditioning (24 hours postoperatively and acute pretreatment). ECG was performed during the whole process of model construction for monitoring cardiac function. Rats in the sham group were subjected to 150 min continuous perfusion. Rats in the I/R and simvastatin groups underwent 30 min ischemia, followed by 120 min reperfusion. Rats in the simvastatin group and I/R group were administrated with 1 mg/kg simvastatin  or 0.1% dimethyl sulfoxide (DMSO) 0.5 h prior to reperfusion, respectively. At 24 h following MIRI, rats were anesthetized for reocclusion of the coronary artery at the initial occluded location, and the heart was collected.Myocardial tissues were extracted after MIRI, homogenized, and cultured. The culture medium was collected for LDH activity determination using the commercial kit . Values at 450 nm were recorded using a microplate reader.w/v) containing 1% Triton X-100 and protease inhibitor [Heart tissues were homogenized in phosphate-buffered saline (PBS) , 515 nm (FITC wavelength), and 560 nm (PI wavelength) were recorded for calculating the apoptotic rate.Homogenized tissues were centrifuged at 1000 rpm for 5 min, and the precipitant was harvested and centrifuged again. Cells were resuspended in 100 Cells were inoculated in a 96-well plate with 3000 cells per well. At the appointed time points, absorbance value at 450 nm of each sample was recorded using the CCK-8 kit  for plotting the viability curves.β-actin as the internal reference. Primer sequences were as follows: HMGB1: 5′-TATGGCAAAAGCGGACAAGG-3′ (F) and 5′-CTTCGCAACATCACCAATGGA-3′ (R); KLF2: 5′-GAGCCTATCTTGCCGTCCTT-3′ (F) and 5′-AGCACGCTGTTTAGGTCCTC-3′ (R); eNOS: 5′-CAACTGGAAAAAGGCAGCCC-3′ (F) and 5′-AAGAGCCTCTAGCTCCTGCT-3′ (R); TM: 5′-CCTTTGTCTTTCCGGGCTCT-3′ (F) and 5′-TCAAGTCCTCCCTACCCTCG-3′ (R); and β-actin: 5′-TGCTATGTTGCCCTAGACTTCG-3′ (F) and 5′-GTTGGCATAGGTCTTTACGG-3′ (R).RNAs were extracted from rat myocardial tissues using TRIzol  and reversely transcribed into complementary deoxyribose nucleic acids (cDNAs) . QRT-PCR was conducted by the SYBR Green method with Tissues were homogenized for extracting proteins. After concentration determination, protein samples were loaded on polyvinylidene fluoride (PVDF) membranes . Subsequently, nonspecific antigens were blocked with 5% skim milk for 2 h. Membranes were then incubated with primary and secondary antibodies. Band exposure and grey value analysis were finally conducted.p < 0.05 indicated the significant difference.Statistical Product and Service Solutions (SPSS) 22.0  was used for all statistical analyses. Data were expressed as mean ± SD (standard deviation). Comparison between multiple groups was done using the one-way ANOVA test followed by the post hoc test (least significant difference). Compared with the sham group, superoxide dismutase (SOD) activity declined and malondialdehyde (MDA) level was elevated in the I/R group. The above changes were markedly reversed by simvastatin treatment Figures . SubsequCell apoptosis is a key event during cardiomyocyte loss and cardiac insufficiency following MIRI. Our findings uncovered that increased apoptotic rate  and decrα (β (α (Hypoxia triggers the deterioration of inflammatory response , 19. Accα , IL-1β (α (β , and IL-α (β  were inhα (β  and MIP-α (β (α  were obsα (β (α .A previous study has shown that simvastatin protects against liver I/R injury through upregulating KLF2 . Here, bMIRI is a pathological state with initial limitation on myocardial blood supply and subsequently reperfusion of blood flow . AlthougCell apoptosis is a determinant event for MIRI-induced myocardial infarction . It is rInflammatory response following MIRI is of significance in aggravating its secondary injury. MIRI may lead to local aseptic inflammation and production of multiple inflammatory factors . HMGB1 iSimvastatin protects inflammatory response at post-MIRI through upregulating KLF2, thus improving cardiac function.


DMD gene encoding diverse isoforms of dystrophin. Loss of full-length dystrophins is both necessary and sufficient for causing degeneration and wasting of striated muscles, neuropsychological impairment, and bone deformities. Among this spectrum of defects, abnormalities of calcium homeostasis are the common dystrophic feature. Given the fundamental role of Ca2+ in all cells, this biochemical alteration might be underlying all the DMD abnormalities. However, its mechanism is not completely understood. While abnormally elevated resting cytosolic Ca2+ concentration is found in all dystrophic cells, the aberrant mechanisms leading to that outcome have cell-specific components. We probe the diverse aspects of calcium response in various affected tissues. In skeletal muscles, cardiomyocytes, and neurons, dystrophin appears to serve as a scaffold for proteins engaged in calcium homeostasis, while its interactions with actin cytoskeleton influence endoplasmic reticulum organisation and motility. However, in myoblasts, lymphocytes, endotheliocytes, and mesenchymal and myogenic cells, calcium abnormalities cannot be clearly attributed to the loss of interaction between dystrophin and the calcium toolbox proteins. Nevertheless, DMD gene mutations in these cells lead to significant defects and the calcium anomalies are a symptom of the early developmental phase of this pathology. As the impaired calcium homeostasis appears to underpin multiple DMD abnormalities, understanding this alteration may lead to the development of new therapies. In fact, it appears possible to mitigate the impact of the abnormal calcium homeostasis and the dystrophic phenotype in the total absence of dystrophin. This opens new treatment avenues for this incurable disease.Duchenne muscular dystrophy (DMD) leads to disability and death in young men. This disease is caused by mutations in the  Duchenne muscular dystrophy (DMD) is a debilitating and lethal neuromuscular disorder. Diagnosis is made usually between the ages of two and five. The earliest symptoms include waddling gate, frequent falling, and difficulties with climbing stairs. However, studies of human foetuses ,2,3 and Progressing skeletal muscle weakness and wasting eventually lead to the loss of ambulation (around the age of 10–12) and to the respiratory impairment, the latter exacerbated by frequent infections. Patients require ventilation at around the age of 20. Cardiac muscle complications are usually the late symptom , suggestAlthough fatal consequences of DMD result from muscle damage, DMD also affects other organs and tissues. The most important are CNS and bone defects, the first leading to cognitive and behavioural impairments, the latter causing skeletal deformities exacerbating muscle symptoms.DMD gene. As the gene locus is on the X chromosome, DMD sufferers are mainly boys, with an incidence of 1 per 5000 male births, but 1 in 50 million females are also affected c) at the level of 100–120 nM, while extracellular [Ca2+] level is approximately 1.0–1.8 mM  [A reduced ability of Camdx mice . The depmdx mice . This maesponse] . Moreoveesponse] ,562+ entry into dystrophic cells is uncertain, given its applicability just to the mechanically challenged cells. Tears are unlikely to occur in dystrophic neurons, yet these cells also show elevated calcium (see below). On the other hand, abnormal shear stress response could perhaps explain elevation of Ca2+ in dystrophic endothelial cells. As mentioned earlier, abnormalities in these cells are puzzling also because of the unclear molecular mechanism. Endothelial cells are not known to express full-length dystrophin and therefore should not be affected by the mdx mutation. Yet, angiogenesis in these mice is seriously altered and impaired vascularisation affects regenerating muscles c correlating with an increased Ca2+ release from intracellular stores, such as ER, was observed in pyramidal cortical and hippocampal neurons c, which could be inhibited by ryanodine, it suggests that Ca2+ release from intracellular stores might be involved [Cognitive impairment in DMD has been associated with changes in the GABAergic system ,107,108.involved . The coninvolved . AlteredMore recently, specific alterations in the expression of glutamatergic and P2 × 7 receptors were identified in the dystrophin-null mice cerebella , a findi2+ overload in neurons and astrocytes, combined with impaired BBB buffering, may be intertwined with the defective synaptic transmission, resulting in the neurobehavioral impairment. Importantly, this dystrophic BBB abnormality is also caused by the absence of Dp427: in mdx glia, Dp71, albeit reduced, was still present [βgeomdx mice [Yet another causative factor in the cognitive impairment might be the dysregulation of the DAP complex, with associated aquaporin isoform 4 (AQP4) water and Kir 4.1 potassium channels, in perivascular glial end-feet. This is associated with a leaky blood–brain barrier (BBB) in dystrophic brains ,111,112.geo mice ,111.2+ concentration within physiological range stimulates mitochondrial energy metabolism and ADP phosphorylation. Thus, calcium homeostasis and mitochondrial energy metabolism are mutually dependent [2+ entry to the mitochondrial matrix is slow because of the low affinity of Ca2+ uniporter (MCU), while Ca2+ removal via mitochondrial Na+/Ca2+ exchanger (NCX) is relatively fast. This prevents mitochondrial Ca2+ accumulation. Therefore, in unstimulated cells, mitochondria do not play a role of calcium stores. However, upon excitation, when a local cytosolic Ca2+ concentration increases, mitochondria may transiently buffer an excess of this cation [2+ uptake by mitochondria under pathological conditions of cellular calcium overload leads to an accelerated ROS generation, opening of the mitochondrial permeability transition pore (MPTP), activation of mitochondria-initiated apoptosis and collapse of the cellular energy metabolism [In all cell types, mitochondria are in the centre of calcium homeostasis. First, they produce cellular ATP, which is necessary to cover the energy demand of calcium transport against the concentration gradient ,113. Secependent ,115. In tabolism ,116,117.2+ uptake ability [2+ accumulation that was observed in dystrophic cardiomyocytes may be considered as a compensatory effect, partially replacing reduced calcium storage capability in the SR/ER system. It has been suggested that more efficient mitochondrial calcium uptake in dystrophic cardiomyocytes may occur because of increased levels of the channel forming subunit of the mitochondrial calcium uniporter (MCU) [+/Ca2+ exchanger are also increased, thus mitochondrial Ca2+ clearance is stimulated. On the other hand, very high cytosolic Ca2+ concentration counteracts calcium extrusion via NCX. Mitochondria in dystrophic cardiomyocytes were speculated to be more resistant to Ca2+-induced damage because of a reduced tendency for opening the mitochondrial permeability transition pore (MPTP) [Interestingly, such disturbances, which are fatal for most cells, in cardiomyocytes might lead to a progressive adaptation of mitochondria, which increase their Ca ability . The eleer (MCU) . Moreovee (MPTP) . The MPTe (MPTP) . Interese (MPTP) ,118.2+ and therefore prone to be opened with increased Ca2+ concentrations. While the amounts of protein components of the pore might be altered, these changes were not consistent [2+ buffering capacity of dystrophic mitochondria was lower than in wild type muscles. Interestingly, a reduced MCU activity in isolated mdx skeletal muscle mitochondria has been found [In dystrophic skeletal muscle, mitochondrial MPTP seems to be more sensitive to Cansistent . Moreoveen found , most lien found . Further2+ release from ER and for its mitochondrial uptake. These contact sites are directly engaged in the modulation of calcium signals and in the activation of mitochondrial energy metabolism. It was found that the density of MAMs in dystrophic cardiomyocytes is increased, while in skeletal muscles it is lower than in their wild type equivalents.Apart from the alterations in the amount and activity of mitochondrial calcium-handling proteins, aberrant intracellular architecture of mitochondrial networks and mitochondria–ER interactions have a huge impact on cellular calcium handling and its abnormalities in dystrophic cells. Mitochondria and the ER membranes form sub-compartments known as mitochondrial associated membranes (MAMs), which contain specific calcium toolkit proteins and are crucial for the regulation of Camdx myoblasts [Aberrant organisation of the mitochondrial and ER networks may result from altered protein profiles at the mitochondria–ER junction and from modified cytoskeletal organisation due to lost interactions between dystrophin and actin filaments. It is plausible that pharmacological modulation of such intra-organelle contacts may be a target for DMD therapy. However, changes in the mitochondrial network architecture were also observed in yoblasts  as well yoblasts ,122, whiNormalisation of calcium handling in dystrophic muscles has long been tested as a potential therapeutic approach which could ameliorate DMD progression, and a number of drugs were tested. As many have been summarised in an excellent review  these exThe calcium channel blockers showed little impact on the DMD pathology , indicat2+ homeostasis those causing inhibition of Ca2+ channels in the sarcoplasmic membrane, stabilization of ryanodine receptors in SR/ER, overexpression of SERCA and reduction of the opening probability of MPTP were found to be of significance [mdx mice with SERCA activator reduced cytosolic Ca2+ levels, restored mitochondrial function, enhanced muscular strength, reduced muscular degeneration and fibrosis. Importantly, this treatment prevented exercise-induced muscular damage in the absence of dystrophin (doi:10.1093/hmg/ddab100).Among drugs which directly influence Caificance ,125. In mdx cardiac cells [Metformin treatment normalised MAMs and modulated mitochondrial metabolism in ac cells . Althougac cells ,128.mdx skeletal muscle cells [Deflazacort, which belongs to the class of glucocorticoids used in DMD therapy as anti-inflammatory, is likely to influence SOCE and affect other calcium toolkit proteins. It was shown to enhance mitochondrial efficiency in le cells . Howevermdx cardiomyocytes in vitro ameliorated the calcium overload. Moreover, in mdx mice in vivo, P188 improved cardiac functions [mdx skeletal muscle but even had some deleterious effects on skeletal muscle functions [An interesting example of an approach aimed at the destabilisation and rupture of the dystrophic sarcolemma is the copolymer-based treatment. Application of these membrane-interacting synthetic molecules, known also as poloxamers or pluronics, to unctions . Howeverunctions ,132. Themdx brains [While there are still many questions regarding the pathomechanism(s) of DMD cognitive impairment, improvements resulting from dystrophin re-expression in x brains  indicateIn this respect, a pathological commonality between muscle and cognitive dysfunctions might be expedient. P2RX7 is one of the main drivers of both DMD muscle damage and inflammation , as wellmdx smooth muscle, in which calcium signalling abnormalities are less severe than in skeletal muscle fibres, gentamycin treatment stimulated RyR expression and restored CICR-based activation of calcium signalling [Gentamycin, later supplanted by the more specific Ataluren, initiate a read through premature termination codons and therefore synthesis of full-length dystrophin. In the dystrophic skeletal muscles, gentamycin improved the direct interaction between voltage-gated channels and RyR, which occurs upon cell excitation. In gnalling . Exon skgnalling . Interesgnalling . Thus, t2+ homeostasis. Some indirect data indicate that changes in calcium signalling in BMD cells should be less severe. For example, only limited impact of BMD on mitochondrial metabolism suggests that mitochondrial Ca2+ buffering capacity as well as ATP delivery are not affected [2+ signals in cardiomyocytes derived from IPSc [2+ load and myotube damage in golden retriever dogs [mdx mice [2+ efflux from SR through the inositol trisphosphate receptor (IP3R) [mdx muscle restored the abnormal Ca2+ entry through channels [As explained in the Introduction, BMD is a milder variant of muscular dystrophy caused by mutations that preserve the reading frame and result in truncated but semi-functional mini- or micro-dystrophins being expressed. The BDM progress, albeit slower than in the case of DMD, may also lead to disability and premature death. Surprisingly, we failed to identify in the literature any studies that directly addressed the effects of BMD mutations on the Caaffected . Furtherrom IPSc ; Dystropver dogs  and in mmdx mice . In yet r (IP3R) . Expresschannels . Thus, eAlthough often attempted, DMD pathology cannot be currently explained by a single mechanism. It is likely that specific, often overlapping, sometimes distinct processes are at play in different tissues and at different stages of the disease. However, abnormalities of calcium homeostasis are the common pathological feature found in both DMD patients and all animal models of this disease and in all dystrophic cells, irrespective whether electrically excitable or non-excitable. Mutations affecting the full-length dystrophin expression are both necessary and sufficient for the calcium phenotype to occur, while the loss of short dystrophins may exacerbate it. However, the role of dystrophin in calcium signalling is not completely understood, which may explain why no treatment targeting calcium pathways has been successful so far.2+ concentration, but the biochemical mechanisms leading to that outcome may have components specific to a particular cell type. The intensity of calcium responses in dystrophic cells results from the aberrant, stimulus-dependent interplay between proteins involved in maintaining calcium homeostasis, and these differ across cells.In dystrophic cells, the net outcome is always the elevated resting cytosolic CaDMD gene mutations lead, in these cells, to significant calcium abnormalities. In fact, already in the developing dystrophic stem cells (including ESC), in mesenchymal and myogenic cells, significant calcium anomalies are not only present but appear to contribute to the early pathology.In skeletal muscles, cardiomyocytes, and neurons, the DAP complex, properly assembled only in the presence of dystrophin, appears to serve as a scaffold for proteins engaged in calcium homeostasis, while dystrophin interactions with the actin cytoskeleton influence ER organisation and motility. However, in some cells, calcium abnormalities cannot be linked to the increased sensitivity to mechanical stimuli or even clearly attributed to the loss of interaction between dystrophin and the calcium toolbox proteins. For example, myoblasts, lymphocytes, and endotheliocytes, despite expressing dystrophin transcripts, are not known to synthesise detectable levels of Dp427 protein. Yet, Given that the impairment of calcium homeostasis seems to underpin multiple DMD abnormalities in a plethora of dystrophic cells, understanding mechanisms behind this alteration may lead to the development of better therapeutic approaches to treat this devastating and incurable disease.


PEITC dose-dependently reversed the ALI-induced increase in plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, and interferon (IFN)-γ and reduced the protein levels of hepatocyte pyroptosis markers such as Nod-like receptor family pyrin domain containing 3 (NLRP3), cleaved caspase-1, and cleaved gasdermin D (GSDMD). In vitro experiments have also verified the inhibitory effect of PEITC on hepatocyte pyroptosis. Furthermore, PEITC inhibits pyroptosis by interacting with cysteine 191 of GSDMD. In summary, our findings establish a role for PEITC in rescuing hepatocyte pyroptosis via direct inhibition of GSDMD, which may provide a new potential therapeutic strategy for ALI.Acute liver injury (ALI), often caused by viruses, alcohol, drugs, etc., is one of the most common clinical liver diseases. Although pyroptosis plays an important role in ALI, there is still a lack of effective clinical drugs related to this mechanism. Here, we show that phenethyl isothiocyanate (PEITC), a natural compound present in cruciferous vegetables, can significantly alleviate concanavalin A (ConA)-induced inflammatory liver damage and carbon tetrachloride (CCl Acute liver injury (ALI) is one of the most common clinical liver diseases and is often caused by viruses, alcohol, drugs, etc. If it is not treated in time, it can easily develop into acute liver failure, chronic liver injury, or hepatocellular carcinoma, which seriously threatens human quality of life and health. The mechanisms and characteristics of ALI are very complex, including hepatocyte death, oxidative stress, inflammation, etc. Hepatocyte pyroptosis is particularly critical for liver damage in the development of liver inflammation into cirrhosis, liver fibrosis, and hepatocellular carcinoma –4. In moPyroptosis is a programmed cell death mediated by gasdermin family proteins , and itsAlthough the liver has a regenerative function, ALI may develop into acute liver failure with extremely high mortality if liver regeneration cannot offset a large number of hepatocyte deaths . When acPhenethyl isothiocyanate (PEITC) is an isothiocyanate compound from the secondary metabolites of cruciferous plants hydrolyzed by myrosinase . Isothiovia its unique structure of mercapten-targeting cysteine 191 of GSDMD.In this study, we discovered that PEITC has a significant hepatoprotective effect, which reverses the increased levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in mice with ALI. Furthermore, this effect of PEITC may be due to inhibition of hepatocyte pyroptosis 4 and olive oil were purchased from Nanjing Chemical Reagent Co., Ltd. . Lactate dehydrogenase (LDH) assay kit, alanine aminotransferase (ALT) assay kit, and aspartate aminotransferase (AST) assay kit were from Nanjing Jiancheng Bioengineering Institute . A mouse TNF-α ELISA kit and IFN-γ ELISA kit were purchased from Dakewe Biotech Co., Ltd. . Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12), Lipofectamine 3000, and Alexa Fluor 488-conjugated anti-rabbit IgG were purchased from Thermo Fisher Scientific . ExFect Transfection Reagent was purchased from Vazyme . Dexamethasone, adenosine triphosphate (ATP), penicillin–streptomycin solution (100×), cell lysis buffer for Western blotting and immunoprecipitation, and RIPA lysis buffer were provided by Beyotime Biotechnology . Insulin-Transferrin-Selenium (ITS) Media Supplement (100×) was purchased from R&D Systems . LPS and ConA were bought from Sigma-Aldrich . Necrosulfonamide (NSA) was purchased from MedChemExpress . Antibodies against β-tubulin, β-actin, and GAPDH were purchased from Abmart . Antibody against Nod-like receptor family pyrin domain containing 3 (NLRP3) was purchased from Cell Signaling Technology . Anti-caspase-1 and anti-IL-1β antibodies were provided by Santa Cruz Biotechnology . Anti-GSDMD antibody was from Abcam .PEITC was from JC (Wuxi) Company, Inc. . CClICR male mice (6–8 weeks old) were purchased from the College of Veterinary Medicine Yangzhou University  and grown in an SPF-grade animal room at 21°C ± 2°C with a 12:12-h day-night cycle. PEITC (molecular mass: 163.24) is fat soluble, so it was dissolved in olive oil to the required concentrations.via the tail vein to induce an acute immune liver injury model. Half an hour after ConA injection, the PEITC (post) group was given PEITC . Eight hours after ConA injection, blood was collected, and serum was separated by centrifugation. The mice were then sacrificed and rapidly dissected, and the livers and spleens of the mice were removed and weighed. In addition, liver and spleen index were calculated. The lesions on the surface of liver and spleen tissues were photographed and recorded. At the same time, a piece of liver tissue was removed. One part was fixed with paraformaldehyde, embedded in paraffin and sectioned, and the other part was stored at −80°C.Sixty ICR male mice were randomly divided into six groups: normal group, ConA-treated group, 7.5 mg/kg PEITC preadministered (pre) group, 15 mg/kg PEITC (pre) group, 30 mg/kg PEITC (pre) group, and 30 mg/kg PEITC postadministered (post) group. In the PEITC (pre) group, mice were given PEITC  for three consecutive days. After the last administration, the ConA-treated group, PEITC (pre) group, and PEITC (post) group were injected with 10 mg/kg ConA 4 model group, 7.5 mg/kg PEITC (pre) group, 15 mg/kg PEITC (pre) group, 30 mg/kg PEITC (pre) group, and 30 mg/kg PEITC (post) group. In the PEITC (pre) group, mice were given PEITC  for three consecutive days. After the last administration, the CCl4 model group, PEITC (pre) group and PEITC (post) group were injected with 0.2% (v/v) CCl4  to induce acute chemical liver injury. Half an hour and 8 h after CCl4 injection, the PEITC (post) group was given PEITC . Sixteen hours after CCl4 injection, blood was collected, mice were euthanized, and liver tissue samples were removed.Male ICR mice were randomly divided into six groups: normal group, CClALT, AST, and LDH activities were detected according to the instructions of the assay kits. The levels of serum TNF-α and IFN-γ were tested according to the instructions of ELISA kits.Total RNA of liver tissues was extracted with TRIzol and then reverse transcribed into cDNA . Real-tig for 10 min. After the protein concentrations of each sample were measured, proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes . Different antibodies were incubated according to the molecular weight of target proteins for Western blotting.Cells or liver tissues were lysed in lysis buffer with protease inhibitor cocktail for 30 min on ice, and the supernatants were collected after cell lysates were centrifuged at 12,000×Paraffin-embedded liver tissue sections were deparaffinized, rehydrated, subjected to antigen retrieval, blocked, and then incubated overnight at 4°C with antibodies against NLRP3, IL-1β, caspase-1, and GSDMD. After washing tissue sections with PBST, slides were stained according to the instructions of the anti-mouse/rabbit universal immunohistochemical detection kit  and finally counterstained with hematoxylin.v/v) CO2 at 37°C. To induce the hepatocyte pyroptosis model, AML12 cells were treated with 100 ng/ml LPS for 3 h, then PEITC  for 1 h, and finally 5 mM ATP for 24 h. The cells were collected for subsequent experiments. HEK293T cells were cultured in DMEM supplemented with 10% FBS and penicillin–streptomycin solution (1×) under an atmosphere of 5% (v/v) CO2 at 37°C.The alpha mouse liver 12 (AML12) cell line and HEK293T cell line were obtained from the National Collection of Authenticated Cell Cultures . AML12 cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), ITS media supplement (1×), 40 ng/ml dexamethasone, and penicillin–streptomycin solution (1×) under an atmosphere of 5% . The mouse GSDMD WT-EGFP plasmid was transfected into AML12 cells, and human GSDMD WT-EGFP or human GSDMD C191A-EGFP plasmid was transfected into HEK293T cells for subsequent experiments.After AML12 cells were incubated with DMSO or PEITC (3 μM) for 2 h, the cells were collected, washed, and resuspended in PBS. The cell suspension was divided into 10 aliquots and heated at the indicated temperatures. Finally, the cell supernatant at each temperature was analyzed by western blot. HEK293T cells transfected with human GSDMD WT-EGFP or human GSDMD C191A-EGFP plasmid were treated with DMSO, PEITC (3 μM) or NSA (3 μM) for 2 h, and the subsequent operations were basically the same as above.After the mouse GSDMD WT-EGFP plasmid was transiently transfected into AML12 cells for 24 h, the cells were lysed with lysis buffer, and the supernatant was collected after centrifugation. PEITC was gradually diluted from 500 μM and added to an aliquot of cell lysis. They were mixed and sucked into Monolith NT.115 capillary tubes . Finally, samples were detected by Monolith NT.115 (Nanotemper Technologies), and data were analyzed by MO.Affinity Analysis. HEK293T cells were transiently transfected with human GSDMD WT-EGFP or GSDMD C191A-EGFP plasmid for 24 h and then lysed with lysis buffer. The supernatant was collected after centrifugation. PEITC or NSA was gradually diluted from 500 μM and added to an aliquot of cell lysis. After samples were mixed, the subsequent operations were the same as above.After the cell samples were fixed with prefixed electron microscopy fixative , they were sent to Wuhan Servicebio Co., Ltd.  to complete the electron microscope scan.t-test or one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test. p < 0.05 was considered significant.The results are displayed as the mean ± SEM, and each experiment included triplicate sets. GraphPad Prism 8 Software  was used to conduct all statistical analyses. Data were statistically evaluated by Student’s The molecular structure of PEITC is presented in Next, we detected the levels of hepatic biochemical indexes ALT, AST, and LDH in the sera of mice from each group. The results showed that the levels of ALT, AST, and LDH in the ConA-treated group were significantly higher than those in the normal group, and PEITC inhibited the increase in ALT, AST, and LDH enzyme activities in the serum of ConA-treated mice in a dose-dependent manner  and GSDMD (GSDMD and GSDMD-N) increased in the ConA-treated group compared with the normal group . The cellular thermal shift assay (CETSA) showed that mouse GSDMD was completely degraded at 64°C in the DMSO group, while PEITC treatment accelerated the degradation of mouse GSDMD at 61°C and reduced the thermal stability of mouse GSDMD  group and PEITC (post) group were restored in a dose-dependent manner, and the activity of serum ALI-related biochemical indicators ALT, AST, and LDH was downregulated .JW, KS, NA, SL, and MB performed research and analyzed the data. JW, KS, and XFW designed the experiments and wrote the paper. XDW, YS, and RD contributed experimental materials and gave suggestions in the formation. JC, XFW, and QX designed the research and obtained funding. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the National Key R&D Program of China (No. 2017YFA0506000), National Natural Science Foundation of China , Natural Science Foundation of Jiangsu Province (BK20191253), and Wuxi Science and Technology Development Fund (CBE01G1650).Author JC was employed by company JC (Wuxi) COMPANY, Inc.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.