We explored the interrelationship between a tissue-specific alternative splicing factor muscleblind-like 1 (MBNL1) and peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α), B-cell lymphoma 2 (Bcl-2) or Bcl-2-associated X protein (Bax) in C2C12 myotubes and mouse skeletal muscle to investigate a possible physiological role of MBNL1 in mitochondrial-associated apoptosis of skeletal muscle. Expression level of PGC-1α and mitochondrial membrane potential evaluated by the fluorescence ratio of JC-1 aggregate to monomer in C2C12 myotubes were suppressed by knockdown of MBNL1. Conversely, the ratio of Bax to Bcl-2 as well as the apoptotic index in C2C12 myotubes was increased by MBNL1 knockdown. In plantaris muscle, on the other hand, not only the minimum muscle fiber diameter but also the expression level of MBNL1 and PGC-1α in of 100-week-old mice were significantly lower than that of 10-week-old mice. Furthermore, the ratio of Bax to Bcl-2 in mouse plantaris muscle was increased by aging. These results suggest that MBNL1 may play a key role in aging-associated muscle atrophy accompanied with mitochondrial dysfunction and apoptosis via mediating PGC-1α expression in skeletal muscle. Skeletal muscle atrophy, which is defined as a loss of muscle mass, is induced by inactivity ,2,3, disSeveral characteristics of sarcopenia are consistent with the muscular symptoms of myotonic dystrophy type 1 (DM1), including insulin resistance, satellite cell senescence, and mitochondrial dysfunction . DM1 is Mitochondrial dysfunction is postulated to be linked with aging-associated impairment of skeletal muscle . PeroxisPGC-1α also plays a role in the regulation of apoptosis. Absence of PGC-1α upregulates Bax expression and increases the ratio of Bax to Bcl-2 . MitochoIn the present study, we investigated the interrelationship between MBNL1 and PGC-1α, Bax, Bcl-2, or mitochondrial membrane potential in C2C12 myotubes and mouse skeletal muscle. We also explored the putative of MBNL1 in mitochondrial-associated apoptosis in skeletal muscle.p < 0.05, −76% in protein: p < 0.05; p < 0.05; Firstly, we investigated the effects of MBNL1 knockdown on differentiation in C2C12 cells. Both mRNA and protein expression level of MBNL1 in C2C12 myotubes was decreased by knockdown of MBNL1 (siMBNL1). There was a significant difference in mRNA and protein expression level of MBNL1 between targeting and scrambled small interfering RNA (siRNA) for MBNL1 . According to the cut-off point  < 0.05 and |log2FC| > 1), 81 differentially expressed genes (DEGs) were identified between targeting and scrambled siRNA for MBNL1. Of these genes, 54 were upregulated and 27 were downregulated. DEGs associated with the membrane or plasma membrane were affected by MBNL1 knockdown . Table 1Ppargc1a gene, which encodes the PGC-1α protein. PGC-1α is considered to be a master regulator of mitochondrial biogenesis [p < 0.05, p < 0.05, p < 0.05, p < 0.05, Among the identified downregulated genes associated with downregulation of MBNL1, we focused on the ogenesis . RT-PCR In the present study we evaluated mitochondrial function in C2C12-derived myotubes by analyzing the changes in the potential of the mitochondrial membrane. This was achieved using the fluorescent probe JC-1 A.p < 0.05, p < 0.05, In healthy cells with high ΔΨM , JC-1 forms complexes known as JC-1 aggregates with intense red fluorescence. Conversely, in cells with low ΔΨM, JC-1 remains in the monomeric form, which exhibits green fluorescence. Consequently, mitochondrial depolarization (a process that occurs in apoptosis ) is indip < 0.05, p < 0.05). p < 0.05, We next turned our attention to animal studies to deepen our understanding of MBNL1. Initially, a comparison of young versus old mice was made. Body weight, absolute muscle wet weight, and muscle weight relative to body weight in young and old mice are shown in p < 0.05, p < 0.05, p < 0.05, p < 0.05, To investigate a physiological role of MBNL1 in aging-associated muscle atrophy, we compared the expression level of MBNL1, PGC-1, Bax, and Bcl-2 in plantaris muscles of young and old mice. Expression levels of MBNL1 mRNA and protein in plantaris muscles of old mice were significantly lower than that of young mice  with the increased ratio of Bax to Bcl-2. Lower expression levels of MBNL1 (mRNA and protein) and PGC-1α (protein) and smaller muscle fiber size in plantaris muscles of old mice were observed compared to young mice. The ratio of Bax to Bcl-2 in mouse plantaris muscle was also increased by aging.In this study, the expression levels of MBNL1 mRNA and protein in C2C12 myotubes were decreased by ~70% and ~76%, respectively, following siMBNL1 treatment. On the other hand, no effects of siMBNL1 treatment on mRNA expression level of MBNL2 was observed. However, MBNL1 knockdown seems to have no impact on the number of nuclei per myotube, myotube diameter, and the expression level of myogenin mRNA and CK protein—these molecules are generally accepted as a differentiation marker . TherefoPpargc1a gene, encoding the PGC-1α protein. The present study also showed that knockdown of MBNL1 is accompanied by a downregulation of PGC-1α at the mRNA level (~68% reduction) and the protein level (~23% reduction). There is no evidence that MBNL1 directly mediates the expression level of PGC-1α. Therefore, MBNL1 may play a role in the regulation of PGC-1α expression. Additional experiments are needed to elucidate this issue.In the present study, RNA-Seq analysis showed that the downregulation of MBNL1 yielded 81 DEGs, 54 of which were upregulated and 27 downregulated. Among the downregulated genes, we focused on the Since PGC-1α plays a key role in mitochondrial function and apoptosis in skeletal muscle , we inve2O2-associated apoptosis [It has been reported that the loss of total protein in C2C12 myotubes caused by Hpoptosis . TherefoIn the present study, the expression level of Bax, the ratio of Bax to Bcl-2, as well as the apoptotic index was increased by MBNL1 knockdown. It has been reported that the ratio of Bax to Bcl-2 is a valid indicator for developing apoptosis in various cells ,39. ThesBalb-c mice were investigated. In this study, on the other hand, MBNL1 expression level in plantaris muscle of C57BL/6J mice was investigated. Differences in mouse strains and/or muscle types may influence the expression level of MBNL1 with aging.In this study, muscle wet weight relative to body weight, muscle fiber size, and the expression level of both MBNL1 and PGC-1α in plantaris muscle of old mice were lower than those of young mice. A previous study  showed hConversely, the expression level of Bax protein and the ratio of Bax to Bcl-2 in plantaris muscle were significantly increased by aging. These results are consistent with the data obtained from our C2C12 experiments and by those previously reported ,13,14,26In conclusion, MBNL1 may play a key role in aging-associated muscle atrophy, a process accompanied with mitochondrial dysfunction and apoptosis.2. At ~90% confluence, the medium was changed to the same amount of differentiation medium consisting of DMEM supplemented with 2% heat-inactivated horse serum containing low glucose  to induce differentiation. The differentiation medium was changed every 2 days and the culture was maintained for 5 days.Cell culture experiments were carried using mouse myoblast-derived C2C12 cells following the methods previously described . C2C12 cThree days after the initiation of differentiation, we transfected RNA oligos into differentiating myotubes using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) as per the manufacturer’s instructions. Details for the transfection of siRNA were previously described . BrieflyIn this study, the number of the sample refers to the number of wells in each experimental condition. All analyses on each sample were carried out in duplicate.i.p.) injection of sodium pentobarbital, and all efforts were made to prevent discomfort and suffering. Male C57BL/6J mice at 10 weeks  and 100 weeks  of age were used in this experiment. In hindlimb skeletal muscle of ~100-week-old C57BL/6J mice, apoptotic rate, which is the relative percentage of terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive myonuclei per total myonuclei, might be ~10% [All experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health  and were approved by the Animal Use Committee of Toyohashi SOZO University (2007001). All treatments for animals were performed under anesthesia with intraperitoneal ( be ~10% . Mice inv/v) Protease/Phosphatase Inhibitor Cocktail . Proximal portions of the frozen left plantaris muscles were homogenized in an isolation buffer of tissue lysis reagent  with 1% (v/v) Protease/Phosphatase Inhibitor Cocktail  with a glass homogenizer. The homogenates were centrifuged at 15,000× g (4 °C for 15 min), and the supernatant was collected. A part of the supernatant was solubilized in SDS sample buffer  at a concentration of 0.5 mg of protein ml-1 and was incubated at 95 °C for 5 min. SDS-PAGE was carried out on 10% polyacrylamide containing 0.5% SDS at a constant current of 20 mA for 120 min, as described previously [The protein expression level of MBNL1, muscle-type creatine kinase (CK), PGC-1α, Bax, and Bcl-2 were evaluated by standard immunoblot analyses, as described previously ,43. The w/v) skim milk with 0.1% Tween 20 in Tris-buffered saline (TBS) at pH 7.5. The membranes were then incubated for 2 h with polyclonal antibodies for MBNL1 , CK , PGC-1α , Bax , and Bcl-2 , and then reacted with secondary antibodies (goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase-linked antibody, Cell Signaling Technology). After the final wash, protein bands were visualized by chemiluminescence , and signal density was measured by Light-Capture (AE-6971) using CS Analyzer version 2.08b . Each sample was investigated in duplicate to at least ensure that results were not influenced by loading errors. The densities of Ponceau S staining were carried out as the internal controls for the transfer process and electrophoresis. Standard curves were constructed during the preliminary experiments to ensure linearity.Following SDS-PAGE, proteins were transferred to polyvinylidene fluoride membrane  at a constant voltage of 100 V for 60 min at 4 °C. The membranes were stained by Ponceau S solution (Sigma-Aldrich) to show equal loading, and blocked for 1 h at room temperature in a blocking buffer: 5%  method using Takara Thermal Cycler Dice Real Time System Software Ver. 4.00 (Takara Bio). To normalize for the amount of total RNA present in each reaction, we used β-actin as internal standard. Primers were designed using the Takara Bio Perfect Real Time Support System (Takara Bio). The following primers were used: MBNL1, 5′-GGACAGCTTGTAGTTTGCCAGGA-3′ (forward) and 5′-GCAGATTTGGCCCAATGGAG-3′ (reverse); myogenin, 5′- CAGTGAATGCAACTCCCACAG-3′ (forward) and 5′- TGGACGTAAGGGAGTGCAGA-3′ (reverse); PGC-1α, 5′-TGATGTGAATGACTTGGATACAGACA-3′ (forward) and 5′-GCTCATTGTTGTACTGGTTGGATATG-3′ (reverse); β-actin, 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ (forward) and 5′-ATGGAGCCACCGATCCACA-3′ (reverse).Total RNA was extracted from C2C12 myotubes treated by MBNL1 siRNA or scrambled siRNA using the miRNeasy Mini kit. The preparation of the cDNA library and the RNA sequencing was performed by Bioengineering Lab . The cDNA originating from the RNA fragments were paired and sequenced using NextSeq 500 .http://david.abcc.ncifcrf.gov/).Tophat2 (ver. 2.1.0) was used to map the sequencing reads to the mouse genome. The sequencing reads were counted using featureCounts (ver. 1.5.0-p3). DESeq2 was used to perform the analysis of differentially expressed genes (DEG). The cutoffs of the DEG approach were chosen as false positive rate (FDR) < 0.05 and |log2 (fold change)| > 1. Gene Ontology (GO) enrichment analysis was performed for DEGs in the gene co-expression network with DAVID  Sequence Read Archive (DRA), and they are accessible through DRA accession number DRA010689.Myotube diameter was evaluated following the methods previously described . BrieflyFrozen plantaris muscles were cut cross-sectionally into halves. Serial transverse cryosections (8 μm thick) of the midbelly region of the proximal side were sliced at −20 °C and mounted on glass slides. The slides were air-dried and stained by using hematoxylin–eosin. The images of muscle sections were incorporated into a personal computer by using a microscope . Minimum diameter of individual fibers  in plant2 incubator at 37 °C for 15 min. Images were then viewed and scanned by a fluorescence microscope , at 470 nm excitation and 525 nm emission for green, and at 560 nm excitation and 630 nm emission for red. Red emission of the dye represented a potential-dependent aggregation of JC-1 in the mitochondria. Conversely, green fluorescence appearing in the cytosol after mitochondrial membrane depolarization represented the monomeric form of JC-1. More than 100 areas were selected from each group. The average intensity of red and green fluorescence was measured using Image J software . The ratio of JC-1 aggregate (red) to monomer (green) intensity was then calculated.Mitochondrial membrane potential in C2C12 myotubes were assessed using JC-1 mitochondrial membrane potential assay kit , according to the manufacturer’s instructions. Briefly, JC-1 solution (100 μL/mL culture medium) was added to the culture medium and was then mixed gently. The cells were then incubated in a COTo evaluate the contribution of apoptosis to MBNL1-associated changes in C2C12 differentiation, we stained cells with Hoechst 33342 to identify apoptotic nuclei. Briefly, a final concentration of 50 μg/mL Hoechst 33342 stain  was added to the culture medium. After 10 min of incubation, the medium was removed, and cells were rinsed in phosphate buffer solution. Analysis was performed immediately under fluorescence microscopy. The nuclei of healthy cells are generally spherical, and the DNA is evenly distributed. During apoptosis, the DNA becomes condensed, but this process does not occur during necrosis. Nuclear condensation can therefore be used to distinguish apoptotic cells from healthy cells or necrotic cells. Hoechst 33342 binds to DNA, and can be used to observe nuclear condensation . To quant-test following the F-test for equal variances. The differences between groups were considered statistically significant at p < 0.05.All values were expressed as means ± standard error of the mean (SEM). Statistical significance was analyzed using an unpaired Student’s


The development of high-throughput gene manipulating tools such as short hairpin RNA (shRNA) and CRISPR/Cas9 libraries has enabled robust characterization of novel functional genes contributing to the pathological states of the diseases. In acute myeloid leukemia (AML), these genetic screen approaches have been used to identify effector genes with previously unknown roles in AML. These AML-related genes centralize alongside the cellular pathways mediating epigenetics, signaling transduction, transcriptional regulation, and energy metabolism. The shRNA/CRISPR genetic screens also realized an array of candidate genes amenable to pharmaceutical targeting. This review aims to summarize genes, mechanisms, and potential therapeutic strategies found via high-throughput genetic screens in AML. We also discuss the potential of these findings to instruct novel AML therapies for combating drug resistance in this genetically heterogeneous disease. Acute myeloid leukemia (AML) is one of the most aggressive forms of hematopoietic disorders. Estimated by the American Cancer Society, there will be about 20,000 new cases of AML and nearly 12,000 deaths from AML in the United States for 2020. Even with intensive chemotherapy and allogeneic hematopoietic stem cell transplantation, the survival outcomes of AML patients remain remarkably low . MoreoveTraditionally, uncovering AML genotype-to-phenotype relationships has been heavily reliant on sequencing clinical samples, identifying AML-associated mutations, and subsequently mutating and/or altering gene expression levels in a laboratory setting to observe a phenotype of interest. Impressively, the number of genes identified as related to AML survival has drastically increased in the past decade, primarily credited to the availability of novel genetic screening technologies such as RNA interference (RNAi) ,6,7 and Since 2006, several research groups have been dedicated to providing genome-wide and pathway-focused libraries for functional genetic screens. For example, The RNAi Consortium (TRC) shRNA library  and the The genome in eukaryotic cells is orchestrated in the form of the chromatin fiber, which has a basic unit, the nucleosome, that consists of four types of histone proteins  surrounded by ~150 base pairs of DNA . ChromatEpigenetic cofactors include enzymes that modify nucleotide bases and histone tails (writers), proteins that recognize these modifications by binding to specific epigenetic alterations (readers), and enzymes that remove the epigenetic modifications (erasers) . The essHistone writers such as lysine methyltransferases (KMTs) and lysine acetyltransferases (KATs) are a subclass of epigenetic writers that modify histones through the addition of methyl and acetyl moieties . KMTs meMOF, also called KAT8, is a member of the MYST acetyltransferase family that is characterized by the presence of the highly conserved acetyl-CoA binding motif (MYST domain)  and is iSETDB1, a SET domain-containing KMT, is a transcriptional repressor via trimethylation of H3K9 b 53,54],5453,54]KAT2A, also known as general control nonderepressible-5 (GCN5), is an enzyme involved in acetylation of H3K9 c and sucHBO1, an MYST acetyltransferase also known as KAT7, specifically acetylates H3K14 d to reguThe epigenetic modifications deposited by writers on chromatin are recognized by nuclear proteins with specific binding domains, known as readers. For example, Bromodomains and YEATS domains recognize multiple types of acetylated histones . On the BRD4, a member of the bromodomain and extra terminal domain (BET) family, recognizes several acetylated positions on H3 and H4 e 61]. B. B61]. BENL, a YEATS domain-containing protein, is one of the MLL translocation partner genes frequently observed in MLL-r leukemia patients ,68. It iThe methyl- and acetyl-modifications made by histone writers can be removed by specific enzymes known as histone erasers. Demethylation of lysine residues relies on two different families: the lysine-specific demethylase (LSD) family and the Jumonji C (JmjC) domain family . The LSDJMJD1C, a predicted histone demethylase at H3K9 f, was a f/f-MLL-AF9 leukemic cells ,9190,91]Increased PRL-3  expression is associated with AML development ,95. HoweCKMT1 is a creatine kinase located in mitochondria that catalyzes the bi-directional conversion between creatine/ATP and phosphocreatine/ADP c, therebLKB1 is a serine/threonine-protein kinase that regulates cell processes such as DNA damage response, cell polarity, and apoptosis ,101. In Gene expression prior to protein translation can be divided into two major steps: gene transcription and RNA processing. Dysregulation of these steps may mislead the expression of cancer-related genes and trigger leukemia development . For exaZEB2, also known as SIP1, is associated with early fetal development through the TGFβ pathway and is known to promote epithelial–mesenchymal transition (EMT) by repressing the expression of epithelial cell–cell junction genes . In a geZFP64, also called ZNF338, interacts with the transmembrane receptor Notch1 in myoblast cells  and is aRBM25, an RNA-binding protein (RBP) that regulates pre-mRNA splicing, is known to mediate cell apoptosis by controlling splicing of the BCL2 family protein BCL2L1 . To explRBM39, also known as RNPC2, is another RBP identified from a library screening of 2900 sgRNAs targeting 490 RNA-binding domains of RBPs in multiple cancer cell models  prominenThe low overall survival rates observed in patients with AML reflect the enormous clinical and molecular heterogeneity of AML . In addiCytarabine, also known as cytosine arabinoside (ara-C), is one of the most effective drugs for AML treatment over the past 40 years . It killWEE1 is a nuclear-localized Ser/Thr-protein kinase involved in the transition between DNA replication and mitosis (S-to-G2 phase transition) through mediating CDK2 activation . WEE1 waTo understand pathways involved in chemo-resistance, a genome-wide CRISPR/Cas9 screen was conducted in AML cells treating with cytarabine . In thisThe FMS-like tyrosine kinase 3 (FLT3) gene is mutated in approximately 30% of AML patients . Since pSPRY3, an antagonist of the fibroblast growth factor (FGF) pathway , was ideATM is a serine/threonine kinase involved in activating the DNA damage checkpoint  and promGLS, the first enzyme catalyzing glutamine to glutamate to enter the TCA cycle , was ideVenetoclax  is an orally bioavailable small-molecule inhibitor that selectively targets BCL-2, a protein involved in antagonizing cellular apoptosis . In 2016Mutation of tumor suppressor TP53  is frequently observed in AML patients (5–20%) . To undeIn a genome-wide CRISPR/Csa9 screen project aimed to identify synthetic vulnerabilities to BCL-2 inhibition in AML, sgRNAs targeting CLPB were found to sensitize AML blasts to venetoclax . This stBET proteins consist of BRD1/2/3/4 and bromodomain testis-specific protein (BRDT). This protein family contains the signature bromodomains that recognize histone acetylation and is involved in transcriptional initiation, elongation, and cell cycle regulation ,129. SinPRC2, an H3K27 methyltransferase complex h, consisKDM1A, also known as LSD1, is a corepressor that mediates demethylation of H3K4 mono- and di-methylation i 132]. . 132]. KThe application of large-scale sequencing efforts has uncovered the highly complicate genetic landscapes in AML patients ; however


Ca2+ entering the mitochondria is necessary for ATP production and the contractile activity of cardiomyocytes. However, excessive Ca2+ in the mitochondria results in mitochondrial dysfunction and cell death. Mitochondria maintain Ca2+ homeostasis in normal cardiomyocytes through a comprehensive regulatory mechanism by controlling the uptake and release of Ca2+ in response to the cellular demand. Understanding the mechanism of modulating mitochondrial Ca2+ homeostasis in the cardiomyocyte could bring new insights into the pathogenesis of cardiac disease and help developing the strategy to prevent the heart from damage at an early stage. In this review, we summarized the latest findings in the studies on the cardiac mitochondrial Ca2+ homeostasis, focusing on the regulation of mitochondrial calcium uptake, which acts as a double-edged sword in the cardiac function. Specifically, we discussed the dual roles of mitochondrial Ca2+ in mitochondrial activity and the impact on cardiac function, the molecular basis and regulatory mechanisms, and the potential future research interest.Calcium ion (Ca A deficiency of cardiac mitochondrial Ca2+ would impair the mitochondrial function and reduce the energy supply, leading to cell damage or death. Besides the energy supply, mitochondria Ca2+ homeostasis is also essential in other biological functions in cardiomyocytes. For example, mitochondria could buffer the concentration of cytosolic Ca2+, which stimulates cardiac excitation-contraction coupling (ECC)  is constantly controlled through two major operative regulatory systems by releasing Ca2+ into the cytosol or up-taking Ca2+ from the cytosol. Voltage-dependent anion channel 1 (VDAC1), located at the outer mitochondrial member, is the first gatekeeper of Ca2+ entering mitochondria [2+ was kept at the mitochondrial intermembrane space (IMS). Then, it enters the mitochondrial matrix through mitochondrial Ca2+ uniporter (MCU). By interacting with a variant of regulatory proteins, such as calcium uptake proteins (MICUs), MCU constitutes a comprehensive functional complex to control the uptake of Ca2+ into the mitochondrial matrix. On the other hand, Ca2+ stored in the matrix can be pumped back to IMS through Na+/Ca2+ (NCX) or H+/Ca2+ exchangers [2+ appear as two independent systems, recent studies indicate that an interlink may exist between the two systems. The uptake of mitochondrial Ca2+ may affect the initiation and activation of the releasing system, particularly mPTP opening under the stress conditions [2+ homeostasis and participate in the cardiac mitochondrial function, such as valosin-containing protein (VCP), a new identified ATPase, which was found to be involved in both mitochondrial calcium uptake and mPTP opening [Despite the importance of mitochondrial Cachondria . Ca2+ wachangers , as wellchangers ,9 , increasing ATP production [2+ could regulate ATPase and ATPase inhibitors to fit the change of cardiac energy demand [As discussed above, mitochondrial Cae minute  and as le minute . When enoduction . It has y demand ,18,19.2+ is released from the sarcoplasmic reticulum (SR) and binds to troponin C, resulting in an interaction between myosin and actin filaments, stimulating ECC leading to cardiac contractions. Mitochondria participate in this process of ECC by buffering the cytosolic Ca2+ and storing Ca2+ in the matrix to generate ATP for the energy required for contraction [2+ concentration could be increased dramatically to 500 to 800 nmol/L compared to 100–200 nmol/L at a normal pace (measured in isolated mitochondria) [2+ upload would impair the heart’s energy production and contractile capability.Moreover, in the cardiomyocytes, Catraction . During 2+ results in oxidative stress and leads to cell death. Cardiac mitochondria produce ROS throughout the respiration chain. Ca2+ promotes ATP production and speeds up the ROS accumulation as well. Excessive ROS induces cardiomyocyte injury, leading to the development of cardiovascular diseases, like ischemia/reperfusion (I/R), myocardium infraction, pressure overload-induced cardiomyopathy and chronic heart failure [2+ can also lead to cardiac dysfunction through other molecules. For example, mitochondrial calmodulin-dependent protein kinase II (CaMKII), a protein found in the mitochondrial matrix, can be activated by both ROS and Ca2+, involving in the mPTP opening under chronic catecholamine stress [On the contrary, excessive cardiac mitochondrial Ca failure ,21. ROS e stress , and proe stress .2+ in chronic heart failure was extensively investigated (as described in Raffaello’s review) [2+ handling was found to be highly associated with heart failure [2+ toxicity in mitochondria are derived from experiments manipulating intracellular Ca2+ homeostasis using genetic approaches. The increasing evidence from these animal models indicated that cytosolic Ca2+ overload induced the impairment of mitochondrial calcium hemostasis, resulting in the opening of mPTP, increased mitochondrial oxidative stress, collapse of mitochondrial membrane potential, impaired ATP production, and necrosis of cardiomyocytes, subsequently leading to heart failure [The impact of mitochondrial Ca review) ,24. The  failure . Numerou failure ,26,27. T failure .2+-induced contractile dysfunction. The first mechanism consists of downregulation of SR Ca-ATPase and upregulation of the NCX function, both tend to shift Ca2+ out of the cell and reduce SR Ca content. This reduces the SR Ca available to be released during subsequent ECC [+ levels. It causes mitochondrial Ca2+ transport out from the matrix through NCX, and impaired Krebs cycles lead to an imbalance between the supply and demand of the energy for the workload [2+ in mitochondria could impact on cardiomyocyte survival and contractility through two primary mechanisms: the reduction of ATP production and the increase of mPTP opening.Three significant mechanisms were revealed to be involved in the Cauent ECC . The secworkload . The thiworkload . Althoug2+ uptake is involved in these two important mitochondrial functions. For example, under physiological conditions, an increase in mitochondrial Ca2+ uptake will enhance the ATP production, which will be beneficial for the cardiomyocyte function. However, in pathological conditions, an increased mitochondrial Ca2+ uptake will induce mitochondrial Ca2+ overload, triggering mPTP opening and resulting in deleterious effects in the cardiomyocytes [ATP generation and mPTP opening are two major events occurring in mitochondria that play the opposite effects in mitochondrial function, as an increase of ATP production will promote cell survival and enhance cardiac functions, an increase of mPTP opening will trigger cell death and impair the cardiac function. Studies have shown that mitochondrial Camyocytes .2+ flux has been identified as the messenger to regulate ATP production. In the 1980s, Denton and McCormack found that Ca2+ can activate the key TCA cycle enzymes: pyruvate dehydrogenase (PDH), oxoglutarate dehydrogenase (OGDH), and can isocitrate dehydrogenase (ICDH) at a micromolar level  [2+-stimulated ATP production by stimulating ATPase. For example, Ca2+ stimulates ATP production by binding to ATPase inhibitor protein (IF1) or ATPase (complex V) itself [2+ regulates ATP production through MCU interacting with a subunit c of ATP synthase [The heart is a high energy demanding organ. Mitochondria occupy a large portion of each myocyte to supply about 90% of the cardiac energy requirement ,32. It ml range) . The act) itself . This cosynthase .2+ with ATP synthase in mitochondria, recent studies also found a dose-dependent association between Ca2+ concentration and ATP production, which may link to the mitochondrial membrane potential (ΔΨ). Using skeletal muscle mitochondria, Sivitz’s group found that the mitochondrial Ca2+ stimulates ATP production at low concentrations (the nanomolar level) while inhibiting the process at a high concentration (the micromolar level) [2+ induced oxidation of NADH which was dependent on complex I substrate: glutamate/malate at a certain respiratory status, the inhibitory effect of high Ca2+ changed ΔΨ independent of the opening of mPTP, suggesting a potential alternative pathway in ΔΨ. In addition, by using adult rats’ cardiomyocytes, Boyman’s group’s results suggested a pathway regulating voltage-dependent ATP production, through which ATP production was regulated by the Ca2+ flux and induced changes in IMM potential as well as the modulation of pyruvate and glutamate dehydrogenase activity by matrix Ca2+ [In addition to the interaction between Car level) . They forix Ca2+ . This re2+ overload. Therefore, it plays a crucial role in the pathological condition, although the molecular structure and regulating proteins are still under investigation. Current reports are debating, for example, while some researches indicate that VDAC at OMM and adenine nucleotide translocase (ANT) at IMM are the structural members of mPTP [2+ treatment, purified ATP synthase activity can be regulated by an mPTP specific agonist and inhibitor, and the purified monomer forms are in voltage-gated and Ca2+-activated channels [The mPTP is a transmembrane protein complex and is located in the IMM. The pore opening can release a substance as big as 1.5 kDa, and it usually opens in response to the matrix Ca of mPTP ,39, the  of mPTP ,40,41,42 of mPTP ,44,45. H of mPTP . Moreove of mPTP . Recentlchannels ,49. A stchannels . Mitocho2+ concentration is one of the factors triggering the pore opening. The consequence of pore opening is a failure in maintaining Ca2+ homeostasis, losing ΔΨ, the rapture of mitochondria, and cell death. Although mPTP opening was considered an alternative way to release matrix Ca2+ and reduce the potentially harmful effect of Ca2+ overload, thus, may play a protective role, the later on ex vivo experiments cannot support this hypothesis [Despite the uncertain regulating mechanisms, it is commonly accepted that a high concentration of matrix Capothesis . More ev2+ uptake is regulated by a multi-protein complex including the main pore unit, MCU and MCU regulatory unit b (MICUb), and its key associate proteins, such as mitochondrial Ca2+ uptake proteins , and essential MCU regulator (EMRE). in MCU, which is critical in pore opening and selectivity filters [2+ uptake, leading to mitochondrial Ca2+ overload and cell death [2+ overload besides affecting the structure of mPTP.MCU presents in almost all cells and is a highly selectively calcium channel responsible for Cachondria . There aake rate . Althougn Nature ,60,61. Sd humans . The tra filters . In huma filters . Althougll death . This st2+ overload-induced cell death is critical to the development and progress of heart failure, and MCU plays an essential role in mitochondrial Ca2+ uptake, researches have been conducted in investigating the impact of MCU in cardiac dysfunction. At least three mouse models with the MCU disruption have been developed by different groups [2+ overload under stress. However, this protective effect was not observed in MCU KO and DN-MCU mice’s hearts [2+ and adjust the energy supply in a Ca2+-dependent manner under stimulation. Interestingly, the mitochondrial Ca2+ concentration in cMCU KO mice is lower than WT at the beginning of catecholamine treatment; it slowly catches up to the level of WT mice. These findings suggested that MCU plays a vital role in the rapid response towards stimulation. Moreover, as both DN-MCU and cMCU KO mice are insensitive towards catecholamine challenges, MCU KO mice act normally [in vitro study showed that overexpressed MCU in mouse primary cortical neurons resulted in more cell death with Ca2+ overload [Since mitochondrial Cat groups , includit groups , an indut groups , and a ct groups . Howevers hearts ,66,68. Inormally ,69,70,71overload . WT hear2+ pass MCU pore, indicating that MICU1 is essential for the Ca2+ selectivity in mitochondria [2+ condition, MICU1 and MICU2 form a heterodimer, and that Glu242 in MICU1 and Arg352 in MICU2 are critical for this bonding. After Ca2+ binds to MICU1 and MICU2, MICU1 and MICU2 bind more tightly and open the MCU pore, and this interaction requires Phe383 in MICU1 and Glu196 in MICU2 [2+-dependent manner. They found that changes in Ca2+ levels affected the interaction of MICU1 and MICU2 with MCU. Specifically, they showed that, at 0.7 to 2 mM Ca2+, the MICU1 dimer showed a reduced binding to MCU, while at concentrations between 1 and 3 mM, the rate of mitochondrial Ca2+ uptake was the highest [2+ level, its effects decreased as cytosolic Ca2+ increased and disappeared above 7 μM. On the other hand, MICU1 has a double role in MCU regulation and is inhibitory at a low Ca2+ level but is activated when the Ca2+ level is more than 2.5 μM [2+ uptake, mitochondrial dysfunction, and impaired energy production at baseline [2+ overload was observed. Instead, the Ca2+ uptake is relatively slow in the MICU2 KO mouse [MICU family members are key regulators of MCU and are relatively well studied compared with the other associate proteins. There are three paralogous  in mice and humans, and the expression level for each of them varies in different tissues . It is kchondria . Evolutichondria , resultiin MICU2 . Petrung highest . In addin 2.5 μM  , the conformational change of the complex weakens the interaction between MICU1-MCU and opens the MCU pore [2+ concentration and dissociates when the Ca2+ concentration is high. The disulfide bond has also been reported between the MICU1-MICU3 heterodimer and MCU in primary cortical neurons [Causing EM . They prMCU pore . This fi neurons .Moreover, it has been found that the protein expression of MICU1 and MICU2 can affect MCU expression. Studies showed that knockdown of either MICU1 or MICU2 could decrease MCU expression . It may 2+ are not fully understood, a few new regulators have been discovered. For example, a study showed that ROS might involve in the regulations of ATP production via the mitochondrial Ca2+ uptake [2+ uptake by interacting with mitochondria [Although the molecular mechanisms underlying the regulation of mitochondrial Ca+ uptake ; SLC25A2+ uptake , and ER chondria .2+ uptake [2+ overload through inhibiting Ca2+ uptake [2+ uptake attenuates the stress-induced reduction of ATP production and the mPTP opening [2+ under stress [2+ in the heart; it also provides evidence supporting a new mechanism of the dual role of VCP-mediated Ca2+ uptake in the ATP production and the mPTP opening.In addition, a recent study has shown that an ATPase protein named valosin-containing protein (VCP) is involved in the regulation of mitochondrial Ca+ uptake . VCP bel+ uptake : an N-te+ uptake . Mutatio+ uptake . Recentl+ uptake ,90,91,92+ uptake . The stu opening . The mecr stress . These s2+ uptake is a double-edged sword regulating cardiac function at both physiological and pathological conditions. In the heart, mitochondrial Ca2+ is essential to maintain contractility, rapidly stimulating ATP production, while mitochondrial Ca2+ overload would induce mPTP opening to lead to cardiac damage and dysfunction. Therefore, maintaining calcium homeostasis in the mitochondria becomes very important in preventing heart disease, as representative studies have shown in 2+ overload.As summarized in 2+, has not been studied. Third, it is also essential to determine the regulation in controlling the balance between the calcium uptake and release systems, such as the cellular regulation and interaction among those components, like the structure of the Ca2+ exporter NCX.One of the potential research interests could be discovering the molecular basis and regulatory mechanism of the mitochondrial homeostasis. First, despite the progress on the studies of the biological function of the MCU/MICUs complex, it is far from fully understood. The current studies have a lot of conflict results and unexplained mechanisms. Further exploring the structural and function and the interaction among these structural proteins will help determine the regulatory control within this complex. Aside from these known proteins, attention should be paid to other potential associated proteins for which the functions are poorly understood, such as EMRE, MCUb, and other unknown proteins. Mainly, the different expressions of MICU isoforms in various tissues, indicating that a potential tissue-specific regulation of mitochondrial Ca2+ uptake with other mitochondrial functions, such as ATP production, mPTP opening, and respiration needs to be further explored. Furthermore, more new regulators involving in these mitochondrial functions, like VCP, and their mechanisms need to be discovered in future studies.Another important research area is to find out the distinct regulatory mechanisms in the physiological and pathological conditions. There might also be a different mechanism involved in the development and the adult cardiac function. In addition, the link between the mitochondrial Cain vitro studies from the cells or isolated mitochondria. These measurements could be different from the physiological condition that occurred in vivo. The detecting techniques need to be developed to better reveal the real-time alterations of the calcium dynamics in vivo.In addition, as we mentioned above, most of our research results were observed from the in vitro studies, therapeutic drugs need to be identified by targeting mitochondrial Ca2+ homeostasis regulation. An mPTP inhibitor has been developed and applied with clinical promise. New therapeutic strategies need to be explored in the future.Finally, based on the animal models and


There is a logical summation that integrates both metabolic plus redox homeostasis because the ATP-sensitive K+ channel (KATP) can only be closed when both ATP and H2O2 are elevated. Otherwise ATP would block KATP, while H2O2 would activate any of the redox-sensitive nonspecific calcium channels (NSCCs), such as TRPM2. Notably, a 100%-closed KATP ensemble is insufficient to reach the −50 mV threshold plasma membrane depolarization required for the activation of voltage-dependent Ca2+ channels. Open synergic NSCCs or Cl− channels have to act simultaneously to reach this threshold. The resulting intermittent cytosolic Ca2+-increases lead to the pulsatile exocytosis of insulin granule vesicles (IGVs). The incretin  amplification of GSIS stems from receptor signaling leading to activating the phosphorylation of TRPM channels and effects on other channels to intensify integral Ca2+-influx (fortified by endoplasmic reticulum Ca2+). ATP plus H2O2 are also required for branched-chain ketoacids (BCKAs); and partly for fatty acids (FAs) to secrete insulin, while BCKA or FA β-oxidation provide redox signaling from mitochondria, which proceeds by H2O2 diffusion or hypothetical SH relay via peroxiredoxin “redox kiss” to target proteins.Pancreatic β-cell insulin secretion, which responds to various secretagogues and hormonal regulations, is reviewed here, emphasizing the fundamental redox signaling by NADPH oxidase 4- (NOX4-) mediated H The emerging concept of redox signaling resulted from ceaseless efforts, and decades of studies of the redox homeostasis of cells and cell organelles. Among organelles, the mitochondrion combines the key roles of the metabolic and redox hub. Prototypical redox signaling is involved in mechanisms of hypoxia-inducible factor (HIF) system-initiated transcriptome reprogramming. Both cytosolic and mitochondrial redox signaling were implicated, e.g., in HIF-1α stabilization, either via the oxidation of iron II to iron III of the reaction center of prolyl hydroxylase domain enzymes (PHD/EglN)  and/or t2O2), which fundamentally determines the 1st phase of glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells, hypothetically by a cooperative closing of the ATP-sensitive K+ channel (KATP) together with ATP elevation  initiated redox signaling  c, which is not triggered by the primary CaV-mediated Ca2+ influx (as for the Ca2+ efflux from ER), but originates from signaling by protein kinase A  cells c c . Moreoves itself ,26.The consensus “canonical” mechanism of GSIS in the past has been an exclusive 100% dependence on the elevated OXPHOS upon glucose intake in pancreatic β-cells. Indeed, pancreatic β-cells were adapted by phylogenesis to serve as a perfect glucose sensor. This is already enabled by the specific isoforms of glucose transporters, GLUT2 in rodents and GLUT1 in humans, which provide equilibration of the plasma glucose concentration with the cytosolic glucose concentration in β-cells ,51. FurtNow we know that this G6P flux is branched into glycolysis and PPP . The par+-dependent isocitrate dehydrogenase 3 (IDH3) are enhanced by the incoming Ca2+ to mitochondria upon GSIS  concentration to artificially depolarize the plasma membrane, the IGV exocytosis only affects those IGVs that are already docked to the plasma membrane, whereas glucose stimulation leads to a new recruitment of IGVs to the plasma membrane c [ATP inhibitor) only transiently elevated these resting free [Ca2+]c, and insulin secretion (GSIS) accounted for <10% of GSIS in wt mice PIs. Nevertheless, KIR6.2KO islets responded to 30 mM KCl by secreting insulin, i.e., when plasma membrane depolarization was achieved in this artificial way. KCl-induced depolarization is used by some researchers to model the 2nd GSIS phase. Incubations of KIR6.2KO islets with 10 mM glucose modulated Ca2+ oscillations, and any Vp depolarization was KATP-independent m is regulated by the Ca2+ influx, mediated by the mitochondrial calcium uniporter (MCU) c oscillations. Those are relayed to somewhat delayed steady-state increases in mitochondrial [Ca2+]m, which reach saturation, i.e., maximum values after a certain time [2+]m oscillations, responding to those ongoing in the cytosol, are then superimposed onto the linearly increasing [Ca2+]m up to saturation. When bursting occurred at 10 mM glucose, the higher frequency of action potential spikes within a burst led to a higher amplitude of the [Ca2+]m increase, i.e., higher saturating values m elevations reached up to 880 nM in wt cells, whereas only 530 nM upon the S100G overexpression, which attenuated GSIS. In contrast, the overexpression of S100G in islet β-cells did not decrease the 1st phase of GSIS, but attenuated the 2nd phase.MCU-deficient β-cells did not exhibit the 2nd part of the increase in ATP/ADP . Note thd OXPHOS . Thus, g2) into diacylglycerol (DAG) and IP3 c increase. Most likely, however, there is usually a synergy between all of these mechanisms (i) to (iii). The general role of second messengers in pancreatic β-cells is described below, while the participation of each pathway in insulin secretion stimulated by various secretagogues is described in later sections.G protein-coupled receptors provide a wide range of cell responses that are mutually interrelated. They activate heterotrimeric G proteins, which typically regulate the production of second messengers or signals via other proteins. Thus, the G protein Gαs increases the generation of cyclic AMP (cAMP), whereas Gαi/o decreases it ,181,182. and IP3 ,184. Gα1 and IP3 . The cla and IP3 . Downstr2+ and bicarbonate. One such sAC is also located in the mitochondrial matrix of pancreatic β-cells c then reaches a threshold, at or above which the RyR Ca2+ channel would be open. Note also that sustained CaV opening partially refills the ER Ca2+ stores, hence counteracting the above effect. The most important effect of EPAC2A is the direct activation of TRPM2, essential for KATP-triggered GSIS, i.e., providing an NSCC-mediated depolarization shift of up to −50 mV c increase due to CaV opening and its relaying to a [Ca2+]m increase should activate sAC. The main effector is most likely the matrix-localized mtPKA , were analyzed in insulin-secreting β-cells using TIRF c that determines the surplus insulin secretion.Incretins (GLP-1 or GIP) massively amplify GSIS by signaling via Gαs proteins, tmAC, cAMP and subsequently via both PKA-dependent and EPAC2A-dependent pathways. As described above, the phosphorylation by PKA increases the sensing range of Kates CaV , the GLUates CaV ,195. Simo −50 mV . Since tcis to trans conformation change of the major SNARE complex (see above). Moreover, the EPAC2A pathway via Rap2-calmodulin kinase II (CaMKII) activates RyR, ensuring Ca2+ efflux from the ER, preceded by CaV opening, thus again contributing to the amplification of IGV exocytosis c oscillations of superimposed waves originating due to the oscillating Ca2+ release from the stores, such as ER, IGVs and/or mitochondria. These events were indeed analyzed by conducting simultaneous electrophysiological and Ca2+ oscillation monitoring, the latter using a proper Ca2+ fluorescent probe(s) at 2 mM glucose but with 200 μM tolbutamide blocking KATP c increase over the elapsed time is much higher than upon the net GSIS, which is not amplified by GLP-1.However, note that the above description represents a simplification, since it is necessary to understand the detailed contribution of GLP1R signaling to the overall dynamics of Caing KATP . Under t(7–36) amide (preproglucagon 78–107) also clearly indicated an increased duration of active and silent electrical activity c bursts phases are intermittent with the interburst phases . These are metabolites of branched-chain amino acids (BCAAs). BCKAs involve three major metabolites, 2-ketoisocaproate  , 2-ketoiThe metabolism of BCKAs is initiated in the mitochondrial matrix by the BCKA dehydrogenase complex , following a similar sequence of reactions to the β-oxidation of fatty acids, termed β-like oxidation. The BCKDH reaction  within t2. Subsequently, the electron-transfer flavoprotein (ETF) accepts electrons from BCKDH as one electron carrier and diffuses up to the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETFQOR) located in the matrix surface of IMM c due to the CaV opening are relayed to the increases in mitochondrial matrix [Ca2+]m m  and consBCAAs were previously found to stimulate insulin secretion in vivo in numerous reports, without a consensus on the mechanism. However, it was recognized that BCAAs can either amplify GSIS or act as secretagogues per se. The latter was confirmed to be based on the oxidative decarboxylation of the respective BCKAs . AdditioThe physiological context and relationships between the BCKA-stimulated insulin secretion and GSIS should be the subject of further studies. One can predict GSIS to be faster and BCKA-stimulated insulin secretion to be delayed from the onset of maximum insulin theoretically given by the net GSIS after a meal rich in saccharides and proteins. In contrast, the most typical situation for BCKA-stimulated insulin secretion can occur during fasting, when there are maximum levels of BCKAs in blood plasma.It is still controversial, as to whether there is fatty acid stimulated insulin secretion (FASIS) at low (insulin non-stimulating) glucose concentrations. Some previous observations suggested that glucose should always be present for FAs to induce any insulin secretion response ,314,337.Of course, one should consider the above-mentioned testing conditions with zero glucose as completely non-physiological. Since FAs trigger the action potential at a low, insulin non-stimulating glucose concentration in pancreatic β-cells , FASIS sNevertheless, it seems plausible that a delayed insulin secretion due to chylomicrons should be expected when even the one-hour-lasting 2nd GSIS phase is nearly complete in rodents. At this time, the inhibition of lipolysis ceases due to the return of blood insulin levels and glycemia back to “fasting” values before the meal. However, the incoming chylomicrons should stimulate at least some response, and these responses would thus occur already at “fasting” glucose concentrations. The GLP-1 amplifying component  may precede FASIS, especially if it still occurs during intermediate glycemia. Whether simultaneous responses to GLP-1 and FAs occur in vivo should also be studied. In conclusion, not only due to molecular mechanistic reasons, but due to the physiological timing, it is crucial to study FASIS experimentally. Of course, in supraphysiological experiments FASIS may overwhelm responses to the other secretagogues, especially when applied intraperitoneally or intravenously or when originating from an experimental high-fat diet .2O2 from the enhanced superoxide formation by the respiratory chain and due to the ETFQOR input of QH2 to it, as with BCKAs. In conclusion, MAG-stimulated insulin secretion via GPR119 is also allowed due to the metabolic component, which can be provided by FAs under conditions of fasting glucose. Nevertheless, MAG-stimulation representing an amplification of GSIS is the end point of the so-called glycerol/FA cycle, described below.Physiologically, one cannot separate FASIS from the concomitant portion of insulin secretion stimulated via 2-monoacylglycerols (MAGs). MAGs are agonists for another metabotropic receptor, GPR119, which provides signaling via Gαs and cAMP ,263. ThuFatty acids appear in pancreatic islet capillaries either bound to albumin, being components of lipoproteins of endogenous sources, or part of postprandial chylomicrons resulting from dietary fat lipids. The latter are rich in triglycerides, which are cleaved locally in pancreatic islet capillaries by lipoprotein lipase . The res+, which only occurs upon GSIS, sirtuins are activated and deacetylate the particular CD36 fraction [Secretory phospholipases A2 might also contribute to the free pool of long-chain FAs in the islet capillaries, in addition to PLC, residing on the plasma membrane and being inherent in GPR40 signaling . In parafraction . As a reShort-chain fatty acids act specifically via the GPR41 metabotropic receptor and contribute to a fine tuning of insulin secretion in both fed and fasting states ,353. SimTheoretically, FASIS may contain one component that depends on the metabolism and a second component that relies on stimulation by the metabotropic receptor GPR40 ,361,362.ATP ensemble. The KATP closure should be ensured by the metabolic component of FASIS, which might, as illustrated in ATP ensemble; and simultaneously or alternatively to this, could activate TRPM2. The GPR40-Gαq/11-PLC-DAG pathway, signaling via PKC, reportedly leads to the phosphorylation and activation of another NSCC channel, TRPC3 [2+ efflux from the ER initiated by the CaV opening [The major pathway downstream of GPR40 acts via Gαq/11, activating PLC, releasing IP3 and DAG. Therefore, the most prominent pathway for FASIS under low glucose conditions should be GPR40-Gαq/11-PLC-DAG-PKC, phosphorylating TRPM4 (TRPM5) channels and activating them, which would aid the necessary shift to the depolarization by the 100% closed Kl, TRPC3 . The rou opening .The GPR40 receptor signaling also activates pathways of the signal-regulated kinase 1 and 2 (ERK1/2) . MoreoveDuring GSIS, and because of the operation of the pyruvate/isocitrate and pyruvate/citrate shuttles, conditions are set to promote FA synthesis. Accordingly, metabolomics studies indicated a palmitic acid increase after the transition from low to high glucose conditions . Note al2O2 serves as a redox signaling. As with the β-like oxidation of BCKAs, FA β-oxidation leads to elevated rates of mitochondrial superoxide formation, hence to redox signaling. The mechanism should be identical to that described above. Even under conditions of low glucose, the ETF/ ETFQOR redox relay to complex I of the mitochondrial respiratory chain plus the ETFQOR system serves as the electron acceptor for dehydrogenases of β-oxidation. Interestingly, GPR40 signaling also activates NOX2 [In general, FA β-oxidation involves mitochondrial flavoprotein dehydrogenases, such as the short-chain acyl-CoA dehydrogenase (EC 1.3.99.2); medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3); long-chain acyl-CoA dehydrogenase (EC 1.3.99.13) and very long-chain acyl-CoA dehydrogenase. They all donate electrons to ETFQOR via ETF, and therefore contribute to the mitochondrial superoxide formation, which after conversion to Htes NOX2 .V channels. This is equivalent to another question: whether the GPR40-receptor component of FASIS is absolutely dependent on the metabolic  component. Since there are action potential spikes due to CaV opening upon GPR40 activation under low glucose conditions [2O2 to trigger the “basic” portion of the IGV exocytosis, while the additional amplification provided by GPR40 should operate via the pathways described above in There is the question of whether the GPR40-receptor-dependent component of FASIS also depends on the opening of Canditions , we shou2O2, hence it is also activated by the intramitochondrial redox signaling originating from FA β-oxidation. As a result, free long-chain FAs entering β-cells via CD36 and those cleaved from intracellular MAGs self-accelerate the GPR40 signaling and hence insulin secretion, after being imported into mitochondria and metabolized by β-oxidation. The activated iPLA2γ cleaves free long-chain FAs from mitochondrial phospholipids. The cleaved long-chain FAs diffuse to the plasma membrane and subsequently stimulate GPR40  to be amplified by the action of mitochondrial phospholipase iPLA2γ, which provides the cleaved mitochondrial FAs to GPR40 in insulinoma INS-1E cells  and in mte GPR40 . It shouV opening with probably prolonged bursting may be a cause, which would induce an additional Ca2+ release from stores such as the ER, IGVs or lysosomes. At the same time, the GPR40-pathway stimulation of IGV exocytosis is likely to be maintained. These speculations are supported by so far the largest amount of insulin being secreted by INS-1E cells when palmitic acid plus 25 mM glucose were added together, relative to either stimulation by palmitic acid alone or GSIS alone [It is not surprising that in the presence of both FA and higher glucose concentrations (those stimulating GSIS when applied without FAs), the fraction of metabolic triggering increases. A more intensive intermittent CaIS alone . These aWe discussed whether high glucose is essential for FASIS as such. Vice versa, a certain minimum level of FA metabolism was found to be required even for GSIS. This view was supported by experiments in which the inhibition of triglyceride lipolysis ,369 atte+. Additionally, glycerol-3-phosphatase produces glycerol and thus regulates glycolysis, the cellular redox state, ATP production and other important branches of metabolism [Indeed, the addition of FAs to β-cells and PIs in the presence of insulin-stimulating glucose amplifies GSIS, while β-cell acyl-CoA levels increase and appear to rapidly esterify glycerol 3-phosphate into lysophosphatidic acid and several different glycerolipids . Note thtabolism . The contabolism ,373. Thetabolism . Note thIn INS-1E cells, about two-thirds of the 1st phase of FASIS was dependent on GPR40 and nearly the same 33% of the maximum FASIS amplitude was observed upon silencing iPLA2γ . The remaining FASIS can be considered to be dependent on the elevation of ATP plus redox signaling from β-oxidation.ATP [ATP closure. As we described in detail above, the metabolism of the remaining long-chain acyl-CoAs leads to superoxide/H2O2 formation, which aids the opening of CaL. In contrast, incoming higher glucose in pancreatic β-cells increases malonyl-CoA [Higher glucose also decreases acyl-CoA levels in pancreatic β-cells . PreviouATP , hence donyl-CoA ,374, whionyl-CoA . Neverthonyl-CoA .One can hypothesize that during fasting the increased FAs bound to albumin in circulation contribute to the ongoing basal secretion of insulin upon fasting . HoweverIn contrast, upon high glucose intake, until chylomicrons arrive, albumin-bound FA levels are minimum, because the lipolysis is simultaneously inhibited in white adipose tissue due to the blockage of adipocyte lipases upon ongoing insulin receptor signaling, and so glucose uptake to adipocytes predominates . The timThe arginine-mediated amplification of insulin secretion was previously reported to be independent of nitric oxide synthase . Arginin2O2 diffusion or on the redox relay mediated by thiol-based proteins that reversibly form S-S bridges and are capable of transferring such a transient oxidation to the target [2O2 diffusion, due to non-specific reactions with random targets within the cytosolic (membrane) milieu. In contrast, the peroxiredoxin oxidized into the first stage can diffuse and transfer the redox signal by oxidation of the distal protein target [Mechanisms of redox signal conduction within the cell can either rely on a simple He target . Peroxirn target . It coul2O2 source then oxidizes peroxiredoxin decamers, which do not escape from its proximity, into their high oxidized states. As a result, the only ones that escape by diffusion are kept in the lowest oxidized stages and can oxidize the proper specific target in a sufficient distance. Experimentally, targets such as a signal transducer and activator of transcription 3 (STAT3) or apoptosis signal-regulating kinase 1 (ASK1) were confirmed to react with peroxiredoxin 2 and 1, respectively, in this way [2O2 are yet to be determined, as discussed in Additionally, the so-called floodgate model could explain the spreading of a redox signal over a larger distance ,15. The this way ,384,385.2O2, its detoxification is ensured by the peroxiredoxin/thioredoxin antioxidant system [Note, however, that for high levels of Ht system ,387. As t system ,10,11. St system ,15. In ct system ,17.ATP-dependent insulin exocytosis and all ionic and regulatory phenomena involved also contribute to the maintenance of the insulin gene expression, hence to the maintenance of so-called β-cell identity [Dor and colleagues hypothesized that repeating Kidentity . Subsequidentity . These aidentity . We coul2O2 through an endogenous thioredoxin reductase–dependent mechanism, a finding that can be masked by the use of nonphysiological bolus delivery of oxidants to the experimental system [2O2 (105 M−1 s−1) [2O2 (Km < 20 μM) and the property of peroxiredoxins to be deactivated through oxidation, it has been suggested that peroxiredoxins are the prime candidates for mediators of H2O2 signaling in addition to detoxification in β-cells [2O2 at GSIS in vivo could also be maintained in balance by the peroxiredoxin/thioredoxin antioxidant system. Oxidative stress that contributes to diabetic etiology occurs when such redox homeostasis is impaired.It has long been assumed that pancreatic β-cells are particularly vulnerable to oxidative stress because of the relatively low expression of several antioxidant enzymes, including catalase, superoxide dismutase and GSH peroxidase ,388. Howl system . Given tM−1 s−1) , the aff β-cells ,386,387.2O2 release at GSIS would instead amplify prediabetes symptoms instead of preventing them. In contrast, we predicted that mitochondria-targeted antioxidants should not harm the physiological redox signaling (except that of BCKAs and FAs) and may be better tolerated when the premature oxidative stress in the matrix at the prediabetic stage is treated in this way [Additionally excessive antioxidant protection may harm basic processes in pancreatic β-cells . For exathis way .The term mitochondrial hormesis, or mitohormesis, has been introduced to describe a defense mechanism observed in oxidant-induced stress-responses by mitochondria . PersistIn addition, putative redox-active compounds, such as dietary antioxidants and olive oil nutraceuticals, are being studied as potential regulators of β-cell redox homeostasis, insulin secretion, blood glucose homeostasis and the development of type 2 diabetes


The family of signal-transducing adapter proteins (STAPs) has been reported to be involved in a variety of intracellular signaling pathways and implicated as transcriptional factors. We previously cloned STAP-2 as a c-Fms interacting protein and explored its effects on chronic myeloid leukemia (CML) leukemogenesis. STAP-2 binds to BCR-ABL, upregulates BCR-ABL phosphorylation, and activates its downstream molecules. In this study, we evaluated the role of STAP-1, another member of the STAP family, in CML pathogenesis. We found that the expression of STAP-1 is aberrantly upregulated in CML stem cells (LSCs) in patients’ bone marrow. Using experimental model mice, deletion of STAP-1 prolonged the survival of CML mice with inducing apoptosis of LSCs. The impaired phosphorylation status of STAT5 by STAP-1 ablation leads to downregulation of antiapoptotic genes, Bcl-2 and Bcl-xL. Interestingly, transcriptome analyses indicated that STAP-1 affects several signaling pathways related to BCR-ABL, JAK2, and PPARγ. This adapter protein directly binds to not only BCR-ABL, but also STAT5 proteins, showing synergistic effects of STAP-1 inhibition and BCR-ABL or JAK2 tyrosine kinase inhibition. Our results identified STAP-1 as a regulator of CML LSCs and suggested it to be a potential therapeutic target for CML. Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder that is caused by hematopoietic stem cells (HSCs) expressing the BCR-ABL fusion oncoprotein. This constitutively active tyrosine kinase influences multiple signal transduction pathways, such as Ras/Raf/mitogen-activated protein kinase (MAPK), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), phosphatidylinositol 3-kinase (PI3K)/Akt, and nuclear factor-κB (NF-κB), leading to peripheral granulocytosis, splenomegaly, and thrombocytosis , 2. SincThese clinical observations indicated that the inhibition of BCR-ABL kinase activity alone cannot eliminate CML stem cells (LSCs) –8. PreviThe family of signal-transducing adapter proteins (STAPs), which contains STAP-1 and STAP-2, has been implicated in various intracellular signaling pathways. STAP-1 was cloned as a c-Kit interacting protein by yeast two-hybrid screening of an HSC library in 2000 . Three yIt is still unknown whether STAP-1 plays a role in CML development, although STAP-1 is expected to exhibit similar functions based on the structural homology between STAP-1 and STAP-2. Given the hematopoietic expression of STAP-1, we hypothesized that STAP-1 might also contribute to the function of LSCs. Here we investigated the role of STAPs in CML using an in vivo experimental mouse model.− CD34+ CD38+ hematopoietic progenitor cells and Lin− CD34+ CD38− HSCs  lentivirus, and evaluated the ability of colony formation . These results alluded to the possibility that the aberrant expression of STAP-1 in LSCs is involved in leukemogenesis of CML and specific features of CML stem cells.We first evaluated STAP-1 and STAP-2 expression in human BM. We collected samples from healthy donors and CML chronic phase (CML-CP) patients, and isolated LinSCs Fig. . InteresSCs Fig. . Our obs− CD48− CD150+). STAP-1 expression decreased according to differentiation stage  cells were comparable in colony-forming assays   Fig. . The sup01) Fig. . These fTo further elucidate the effects of STAP-1, we performed a gene expression analysis of WT and STAP-1 KO CML LSCs. Putative differentially expressed genes were identified by RNA-sequencing . There was a total of 3792 differentially expressed genes between WT and STAP-1 KO LSCs, among which 1256 (33.1%) genes were upregulated, while 2536 (66.9%) genes were downregulated Fig. .Fig. 5STp < 0.0001) in STAP-1 KO CML LSCs using Ingenuity Pathway Analysis. We applied z-score value ≥ 2.0 for activated pathways and z-score ≤ −3.0 for inhibited pathways. A total of 76 pathways were significantly altered in STAP-1 KO CML LSCs  using shRNA. The interference of STAP-1 expression was confirmed using quantitative real-time PCR Fig. . AlthougWe next examined the effects of STAP-1 in cultures treated with JAK2 inhibitor, ruxolitinib, since JAK2-STAT5 pathway has been reported to be involved in development of CML LSC –21, 38. Efficient eradication of LSC by the inhibition of BCR-ABL signaling using PPARγ agonist, pioglitazone, has been previously reported . The tre+ CML cells showed that the deletion of STAP-1 had the additive effects with imatinib in two out of six patients  were generated with a targeted disruption in the STAP-1 gene locus . The conFlow cytometric analysis and sorting were performed using standard multicolor immunofluorescent staining protocols with a FACS Aria IIu (BD Biosciences). The indicated antibodies used for flow cytometric sorting and analysis were listed in Supplementary Table 5 BM cells from C57BL/6SJL mice. Four months after the first transplantation, serial transplantation was performed by transferring 2 × 106 BM cells from primary recipients.In all, 5000 (LSK) cells sorted with FACS Aria were transplanted into lethally irradiated C57BL/6SJL mice, together with 5 × 10The cDNA encoding human BCR-ABL was cloned into the EcoRI site of the pMSCV-ires-GFP vector to generate retrovirus particles . Constit4) were transplanted intravenously into lethally irradiated C57BL/6SJL mice together with 3 × 105 BM cells from C57BL/6SJL mice. For serial transplantation, 1 × 106 GFP+ BM cells or 5 × 104 GFP+ LSK cells were isolated from recipient mice 11 days after the first transplantation, and transplanted into lethally irradiated recipient mice together with BM cells from C57BL/6SJL mice.The transduction of BCR-ABL to LSK cells was conducted as previously described . After iCells were cultured in MethoCult medium . The numbers of colonies were counted after 7 days. For serial replating assays, cells were harvested from the first plates and replated in fresh methylcellulose medium. The numbers of colonies were counted again after 7 days.+ cells were assessed with flow cytometry. For Ki67 staining, cells were fixed and permeabilized before staining with APC conjugated anti-Ki67 antibody.To determine the cell cycle status in vivo, mice were administered BrdU intraperitoneally  12 h before analysis. Numbers of BrdU+ cells were stained using PE Annexin V apoptosis Detection Kit (BD Biosciences). For TUNEL staining, GFP+ LSK cells were attached to glass slides by cytospin, fixed with 4% paraformaldehyde, and stained according to the manufacturer’s protocols (Sigma-Aldrich).Isolated GFP+ CD45.2+ LSK cells from WT and STAP-1 KO CML mice were sorted by FACS, and total RNA was extracted with an RNeasy Mini Kit  according to the manufacturer’s instructions. Each cDNA was generated using a Clontech SMART-Seq v4 Ultra® Low Input RNA Kit for Sequencing (Takara Clontech). Each library was prepared using a Nextera XT DNA Library Prep Kit (Illumina) according to the manufacturer’s instructions. Whole transcriptome sequencing was applied to the RNA samples with use of on an Illumina HiSeq 2500 platform in a 75-base single-end mode. Sequenced reads were mapped to the mouse reference genome sequences (mm10) using TopHat ver. 2.0.13 in combination with Bowtie2 ver. 2.2.3 and SAMtools ver. 1.0. The number of fragments per kilobase of exon per million mapped fragments was calculated using Cuffnorm ver. 2.2.1. Access to raw data concerning this study was submitted under Gene Expression Omnibus accession number GSE127984. The data sets were analyzed using Ingenuity Pathway Analysis (Ingenuity Systems Inc).GFP+ CD45.2+ LSK cells from CML mice using the RNeasy Mini kit and converted to cDNA by High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Real-time quantitative PCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) and Taqman gene-specific primers. Primers and probes are reported in Supplementary Table Total RNA was isolated from GFP4/well) in 96-well plates were cultured for the indicated periods. CCK-8 reagent was added and incubated another 4 h at 37 °C and Optical Density was measured at 450 nm with a microplate reader. In some experiments, imatinib mesylate, pioglitazone (Sigma-Aldrich), or ruxolitinib (ChemScene) was added in the cultures.Human CML cell lines  were obtained from the JCRB Cell Bank , and maintained in RPMI  supplemented with 10% fetal bovine serum. For cell proliferation and viability assay, the Cell Counting Kit-8 (Sigma-Aldrich) was used as recommended by the manufacturer. In brief, the cells (1–2 × 10+ cells (4 × 106 cells/ml) were infected by spinoculation . Two adjuvants, poloxamer 407  and prostaglandin E2 , were added to the culture medium [Lentiviruses were produced by transfection in 293T with MISSION shRNA vectors (Sigma-Aldrich). CML CD34e medium . Cells wStable STAP-1 knockdown CML cell lines were established by transfection with MISSION shRNA lentiviral vectors (pLKO.1-puro) targeting human STAP-1 and selection with puromycin . For control, we used MISSION pLKO.1-puro empty vector (#SHC001).The cells were harvested and lysed in lysis buffer . The immunoprecipitates from cell lysates were resolved on SDS-PAGE and transferred to Immobilon filters (Millipore). Immunoblotting was performed with appropriate antibodies. Immunoreactive proteins were visualized by an Odyssey infrared imaging system (LI-COR Biotechnology). Antibodies against c-Abl , Myc , STAT5a , STAT5b , and JAK2  were purchased from Santa Cruz Biotechnology. Antibodies against STAP-1  were purchased from Sigma-Aldrich.t test. Survival curves were calculated using the Kaplan–Meier method and analyzed with the long-rank test. Statistics were analyzed using JMP Pro 13.0.0 software (SAS Institute Inc). No samples were excluded from the analysis. The data had a normal distribution, and the variance was similar between the groups being compared. No statistical method was used to predetermine sample sizes. Sample size was based on previous experience with experimental variability. The experiments were not randomized. The investigators were not blinded to allocation during experiments or outcome assessment.Statistical differences were determined using the Student’s Supplementary figures and tables


We investigated the role of Ca2+ signaling in the PL-driven endothelial healing process. In our experiments, the functional significance of Ca2+ signaling machinery was highlighted performing the scratch wound assay in presence of different inhibitors or specific RNAi. We also pointed out that the PL-induced generation of intracellular ROS (reactive oxygen species) via NOX4 (NADPH oxidase 4) is necessary for the activation of TRPM2 and the resulting Ca2+ entry from the extracellular space. This is the first report of the mechanism of wound repair in an endothelial cell model boosted by the PL-induced regulation of [Ca2+]i. Wound repair is a dynamic process during which crucial signaling pathways are regulated by growth factors and cytokines released by several kinds of cells directly involved in the healing process. However, the limited applications and heterogeneous clinical results of single growth factors in wound healing encouraged the use of a mixture of bioactive molecules such as platelet derivatives for best results in wound repair. An interesting platelet derivative, obtained from blood samples, is platelet lysate (PL), which has shown potential clinical application. PL is obtained from freezing and thawing of platelet-enriched blood samples. Intracellular calcium (Ca Wound healing is a well-orchestrated process strictly dependent on correct functions of several cell types, such as inflammatory cells, endothelial cells, fibroblasts, and keratinocyte cells, with the ultimate goal of restoring tissue integrity . This process relies on a complex integration of some cellular signaling, requiring multiple growth factors and cytokines stimuli, with positive and negative effects on different wound phases .Some of these factors are well characterized, but until now, few of them are available for clinical use due to their short half-life and excessive cost. In particular, the use of single growth factor is not effective in affecting wound repair, and the best solution should be to deliver a mixture of several growth factors in order to induce complete repair ,4.Platelets are important elements in wound repair and tissue regeneration processes, affecting cell recruitment and extracellular matrix remodeling, as well as blood vessel growth and angiogenesis.Platelets are characterized by the presence of granules containing growth factors, cytokines, and chemokines that are able to stimulate cellular activities at wounded sites .An increasing amount of research has shown the positive role of growth factors, released by platelets, if delivered in wound healing sites, stimulating the healing process in a physiological fashion .An attractive platelet derivative, showing potential clinical application, is platelet lysate (PL). PL is obtained from repeated freezing and thawing of platelet-enriched blood samples . This PLWe have previously demonstrated by in vitro scratch wound, cell migration, and microscope image analysis that PL activates the repair mechanisms in various types of injured endothelial cell layers .Our observations suggested that the PL-induced wound healing is driven by a complex of effects, including both cell proliferation and migratory behavior. These data confirm previous findings about the chemotactic responses to platelet derivatives of glial cells , rat fet2+ is essential for cell proliferation and motility i is a proposed trigger in wound healing, and Ca2+ waves spread from injured areas leading to temporary Ca2+ increase. Moreover, a correlation between Ca2+ dynamics and wound repair has been reported in studies on several cell types, i.e., keratinocytes, fibroblasts, and endothelial cells, where PL has induced significant Ca2+ signals, and the use of Ca2+ chelator BAPTA-AM ethane-N,N,N′,N′-tetraacetic acid tetrakis-acetoxymethyl ester) in experiments of scratch wound healing suppressed the healing effect of PL i under 0Ca2+ conditions, which reflects the fundamental role of extracellular Ca2+  is highly expressed in endothelial cells where it mediates redox-activated Ca2+ entry . Hence, 2+]i increase caused by 20% PL exposure  with 10 µM econazole, a TRPM2 inhibitor , was ablexposure A, determse to PL B. The dese to PL C determiexposure D,E.It is commonly known that some growth factors (GFs), such as PDGF  and VEGF2+]i increase caused by 20% PL exposure was completely abrogated  that leads to PIP2 hydrolysis in InsP3 and DAG. It is well known that GFs binding to their specific receptors, belonging to G-protein-coupled receptors (GPCRs) and tyrosine kinase receptors (TKRs) families, determine an increase in [Ca2+]i in response to PL. For this purpose, we utilized a panel of inhibitors, i.e., thapsigargin, 2-APB, U73122, and caffeine.For this reason, we focused on the contribution of the ER in the variation of i increase triggered by PL exposure , a non-competitive inhibitor of the sarco/endoplasmic reticulum Caexposure A,B. 3R i , a PLC inhibitor , determi2+]i A,B. 2+.Cell incubation with vehicle DMSO used alone is ineffective on Ca2+ pool reduction in the ER, following to InsP3-dependent Ca2+ release, led to the activation of store-operated Ca2+ entry (SOCE) [2+]i (The Cay (SOCE) ,29. Thery (SOCE) ,31, causE) [2+]i A,B. 2+ refilling in bEND5 cells i level, characterized by a 100 sec oscillations peak phase, followed by a decrease and stabilization phase also featured by the presence of less intense and less frequent oscillations. This increase was completely prevented by using different inhibitors. In this study, we investigated the role of Ca2+ in the extracellular space, the PL-induced signaling cascade was not triggered, but reestablishment of basal [Ca2+]e generates again a Ca2+ response observable by the presence of two spikes. These findings demonstrate that the onset of the Ca2+ increase after PL addiction depend on Ca2+ influx from extracellular space that is required to sustain the intracellular Ca2+ signal.We observed that in absence of Ca2+ influx plays a pivotal role the TRPM2 channel that is well known to be activated by intracellular ROS. PL, due to its growth factors, is able to generate ROS in the intracellular space. GFs, after binding to their specific receptors, activate the NADPH oxidase complex that led to an increase of intracellular ROS, as we observed with DHR-123 fluorescent probe.In this Ca2+ entry. The existence of the link between PL, TRPM2, and intracellular ROS was confirmed by pharmacological inhibition through apocynin and by RNAi for NOX4, which lead to a reduction of Ca2+ release from the ER is fundamental to sustain the PL-induced cytosolic Ca2+ increase. Our data suggest that depletion of Ca2+ in the ER after pretreatment with thapsigargin determined the complete abrogation of cytosolic Ca2+ rise. Further, Ca3-mediated Ca2+ release showed a pivotal role in PL-induced endothelial tissue regeneration: the inhibition of PLCγ using U73122 i. Such observation was confirmed by using the Mn2+-quenching technique i boosting wound repair in endothelial cell model. We can summarize our results as described in the following model  inhibition of TRPM2 inhibited the Ca2+ response to PL.Data showed that PL could trigger intracellular Ca2+ from the ER through InsP3R, confirmed by the pharmacological inhibition of the PLC/InsP3 cascade. Therefore, the pharmaceutical inhibition of Orai1 also diminished the Ca2+ response to PL, suggesting the involvement of SOCE in sustaining of response to PL. At the same time, GFs binding to their receptors induces PLC activation and consequently the release of CaMoreover, our findings can also represent an important knowledge for the use of PL as an alternative for ex-vivo expansion of endothelial cells before clinical administration, avoiding issues related to the use of fetal bovine serum. All reagents were from Sigma-Aldrich, unless otherwise indicated. 2 in DMEM , supplemented with 10% FBS, L-glutamine (200 mM), 100 U/mL penicillin, and 100 mg/mL streptomycin [The bEND5 cell line  is an immortalized mouse cell line from brain capillary endothelial cells. Cells were grown at 37 °C, 5% COptomycin . 9 cell/mL, was washed three times in Ham’s F10 medium, 0.3% ethylenediamine tetraacetic acid (EDTA) to remove possible traces of plasma factors. To complete PL preparation, the concentrated washed platelets were then subjected to thermal shock: frozen (−80 °C) and thawed (37 °C). Platelet bodies and debris were eliminated by centrifugation and the supernatant was stored in aliquots at −80 °C until use [Platelet concentrates were obtained following a standard clinical method. A platelet-enriched fraction was purified from platelet concentrate obtained according to standard procedures for the preparation of blood components. To obtain PL, the platelets concentrate, at the density of 1 × 10 frozen −0 °C and ntil use ,38,47.Scratch wounds were made in confluent monolayers by using a sterile 0.1–10 µL pipette tip. After washing away suspended cells, cultures were refed with medium in the presence of PL for 24 h. After cell exposures, cells were fixed for 30 min in 3.7% formaldehyde prepared in PBS for 30 min, and then stained for 30 min at room temperature with 0.1% toluidine blue for 30 min. The wound space width was measured at wounding and at the end of treatments, using an inverted microscope  equipped with a digital camera. Digitized pictures of wounds were analyzed using the NIH Image J software. Wound closure was determined as the difference between wound width at 0 h and at 24 h.Cells were lysed in RIPA buffer (supplemented with a protease and phosphatase inhibitor cocktail) and homogenates were solubilized in Laemmli buffer. Amounts of 25 μg of proteins were loaded on gel, subjected to SDS-PAGE (12% gel), and then transferred to a nitrocellulose membrane, using a Bio-Rad Mini Trans Blot electrophoretic transfer unit . Membranes were blocked with 5% nonfat dry milk in PBS and then probed at 4 °C overnight, with a primary antibody against NADPH oxidase (NOX)–4 . Membranes were then washed three times with PBS-0.05% Tween-20 to remove unbound antibodies and further incubated with appropriate horseradish peroxidase-conjugated secondary antibodies . Membranes were developed using an enhanced chemiluminescence kit  according to the manufacturer’s protocol and digitized with Quantity One Image Software . Equal loadings were confirmed with an anti-actin antibody .Intracellular levels of ROS were evaluated using dihydrorhodamine (DHR) 123, a fluorescent dye precursor, which is converted to fluorescent rhodamine 123 upon reaction with ROS. 2, 1 MgCl2, and 5 KCl, pH 7.4. After probe loading and washing, cells were observed through confocal time-lapse analysis, using a Zeiss LSM 510 confocal system interfaced with a Zeiss Axiovert 100 M microscope . Cells were plated on glass-base dishes , allowed to settle overnight, and loaded for 30 min at room temperature in the dark with DHR-123 (30 µM) in a loading buffer consisting of (mM) 10 glucose, 10 Hepes, 140 NaCl, 2 CaClExcitation was acquired by the 488 nm line of an Ar laser, and emission was collected using a 505–550 bandpass filter. ROS production data were expressed as fluorescence arbitrary units [2, 5 KCl, and 1 MgCl2. For Ca2+-free experiments, the ion was omitted from the loading buffer [Cells were plated on glass-base dishes , allowed to settle overnight, and then loaded with Fluo-3/AM (20 mM), a cell-permeant, fluorescent calcium probe in the dark for 30 min at 37 °C. The loading buffer consisted of (mM) 140 NaCl, 10 glucose, 10 HEPES pH 7.4, 2 CaClg buffer ,49,50.After probe loading and washing, cells were analyzed through confocal time-lapse analysis, using a Zeiss LSM 510 confocal system interfaced with a Zeiss Axiovert 100 M microscope . Excitation was realized by the 488 nm line of an Ar laser, and emission was collected using a 505–550 bandpass filter. Kd = 400 nmol/L. Several cells were viewed together through a 20× Plan-Neofluar Zeiss objective (0.5 NA). Fluo-3/AM fluorescence was evaluated in digitized images as the average value over defined contours of individual cells. Fluo-3 calibration was achieved by the following equation :Ca2+ = KFmax and Fmin are maximum and minimum fluorescence intensities obtained by Fluo-3/AM calibration after cell exposure to 500 µM A23187 for about 10 min, followed by addition of 20 mM EDTA.Cells were settled in multi-well plates for 24 h and then subjected to the indicated experimental conditions. NucleoSpin RNAII Kit  was utilized to extract total RNA. Complementary DNA was synthesized from RNA using the Transcriptor First Strand cDNA Synthesis Kit .Ct method. qRT-PCR was performed using Power Sybr Green Mastermix  and KiCqStart® SYBR® Green Primers  and 5’-UCCCAUAUGAGUUCUG-3’ (antisense)) or with equimolar scramble siRNA by using the N-ter Nanoparticle siRNA Transfection System (Sigma-Aldrich). For TRPM2, RNAi was performed using esiRNA . Scramble siRNA was obtained using commercial non-targeting siRNA . Cells were collected at 24 h after transfection and used for the experiments.All statistical tests were carried out with GraphPad Prism 5 .


Dormant chemotherapy-resistant leukemia cells can survive for an extended period before relapse. Nevertheless, the mechanisms underlying the development of chemoresistance in vivo remain unclear.Using intravital bone imaging, we characterized the behavior of murine acute myeloid leukemia (AML) cells (C1498) in the bone marrow before and after chemotherapy with cytarabine.Proliferative C1498 cells exhibited high motility in the bone marrow. Cytarabine treatment impaired the motility of residual C1498 cells. However, C1498 cells regained their migration potential after relapse. RNA sequencing revealed that cytarabine treatment promoted MRTF-SRF pathway activation. MRTF inhibition using CCG-203971 augmented the anti-tumor effects of chemotherapy in our AML mouse model, as well as suppressed the migration of chemoresistant C1498 cells.These results provide novel insight into the role of cell migration arrest on the development of chemoresistance in AML, as well as provide a strong rationale for the modulation of cellular motility as a therapeutic target for refractory AML. Chemotherapy with metabolic antagonists, such as cytarabine (cytosine arabinoside), has been widely used for the treatment of acute myeloid leukemia (AML) for more than 40 years –3. NeverOn the other hand, another study suggested that AML cell migration did not depend on interactions with ligands found in the BM stroma . These cHerein, we provide further insight into the behavior of chemoresistant AML cells in vivo with evidence from intravital time-lapse bone imaging. We further identify the key molecular mechanisms of migration arrest and chemoresistance development in AML cells.C57BL/6 J mice were purchased from Crea Japan Inc. . Female mice (8–10 weeks of age) were used unless stated otherwise. The number of animals used per group in each experiment is provided in the corresponding figure legend. All mice were maintained under specific pathogen-free conditions, and the Institutional Animal Care and Use Committee of Osaka University approved all procedures involving animals.l-glutamine; Nakalai Tesque, Inc.) supplemented with 10% fetal bovine serum (FBS). Successfully transduced cells were selected by cell sorting and expanded as single-cell clones. One clone was selected for each transfected cell line (C1498-EGFP). C1498-EGFP cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.C1498 cells were purchased from American Type Culture Collection. The green fluorescent protein (GFP) coding sequence was cloned in a pMYs retroviral vector . The recombinant virus was produced using the Platinum-E retroviral packaging cell line, Ecotropic , which was transfected using PEI MAX . C1498 cells were stably transduced using the recombinant retrovirus, grown in DMEM  in C57BL/6 J mice.The AML syngeneic mouse model was generated by intravenously injecting 2.0 × 10AML mice were treated with high-dose cytarabine chemotherapy. Twenty micrograms of cytarabine  dissolved in 200 μL PBS was injected intraperitoneally at days 19 and 20 after C1498-EGFP cell injection for intravital imaging; for survival analysis, chemotherapy was administered at days 14 and 15. In addition to cytarabine, some mice also received CCG-203971. For imaging, 200 μg of CCG-203971 dissolved in 50 μL DMSO solution was intraperitoneally injected at day 18, and twice per day at days 19 and 20. For survival curve analysis, 200 μg of CCG dissolved in 50 μL DMSO was administered at day 13, and twice per day at days 14 and 15.Griffonia simplicifolia, Alexa Fluor® 594 conjugate .Intravital two-photon imaging was performed as previously described –17. ImagThe distance between the AML cells and the bone or blood vessels was analyzed using the Imaris software. AML cells were defined as spots with a diameter of 10 μm using the 3D images. The surface of the bones and blood vessels was identified in the same 3D image and transformed to distance gradients. Subsequently, the intensities of the distance gradients of the spots were calculated. We analyzed the displacement area of AML cells by calculating IoUs (intersection over union). Initially, all frames (green channels) of MIP images from each experiment were combined, and thresholds were calculated by the Otsu method. These thresholds were used to distinguish cellular regions from the background. Denoising of the resulting binary images was performed by applying median filters. Finally, we calculated the IoUs between the cell regions of successive frames. To calculate the mean migration speed, we defined AML cells as estimated spots with a diameter of 10 μm from the MIP image; the spot trajectories were automatically analyzed.+ CD49b+ cells were isolated as AML cells using an SH800 cell sorter (Sony).First bone marrow cells were collected from femur, tibia, and pelvis from three untreated and three cytarabine-treated mice 21 days after AML cell injection. The bone marrow cells were blocked with anti-CD16/32 antibody  for 5 min and stained with PE conjugated anti-CD49b antibody  for 30 min. GFPP values (threshold of 0.05) and z-scores were used to identify significant upstream regulators. P value indicated significance, while z-scores were used to define activation (z-score ≥ 2.0) or inhibition (z-score ≤ −2.0). Access to raw data concerning this study was submitted under Gene Expression Omnibus (GEO) accession number GSE149853.Total RNA was extracted using QIAzol lysis reagent (Qiagen) according to the manufacturer’s instructions. Sequencing was performed on an Illumina HiSeq 2500 platform (Illumina) in a 75-base single-end mode and the Illumina Casava software  was used for base calling. Sequenced reads were mapped to mouse reference genome sequences (mm10) using TopHat (v2.0.13) in combination with Bowtie2 (v2.2.3) and SAMtools (v0.1.19). The fragments per kilobase of exon per million mapped fragments were calculated using Cufflinks (v2.2.1). Upstream regulator analysis and causal network analysis was performed using Ingenuity Pathway Analysis  to identify molecular networks within the Ingenuity Knowledge Database. t tests. One-way analysis of variance (ANOVA) was used for comparisons among three groups, while Kolmogorov–Smirnov test was used for comparisons between two groups. Fisher’s exact test was used to calculate P values in IPA upstream analysis. Statistical significance in survival data was determined using the log-rank test. All the statistical analyses (except for RNA-Seq data) were performed using GraphPad Prism 7 (GraphPad Software).Numerical data are shown as a dot plot. Data are expressed as means ± SEM. Statistical significance between groups was determined using two-tailed To establish an AML syngeneic mouse model, we transplanted C1498 murine AML cells intravenously into wild-type C57BL/6 J mice . Prior tIntravital imaging of the parietal BM revealed a constant movement of AML cells along the blood vessels during all disease progression stages Fig. ; Video 1Additional file 1: Video 1. Intravital time-lapse imaging during different phases of progression of AML. AML cells were transplanted into wild-type C57Bl6 mice. AML mice were subjected to intravital time-lapse BM imaging during different disease phases. Green, EGFP-expressing AML cells; red, blood vessels (Alexa Fluor 594); blue, bone tissues (SHG). Scale bar, 50 μm.To compare the dynamics of AML cells between the cytoreductive and proliferative phases after cytarabine treatment, we performed intravital time-lapse imaging on days 21 and 28 (Video 2). AML cell migration is characterized by amoeboid movements, which are driven by repetitive cycles of protrusion and contraction. Thus, we classified AML migration into two types: a constantly changing cell shape characterized by protrusions or migration driven by extensive contraction and protrusion. To quantify the extent of cell shape alterations, we calculated the “Intersection over Union (IoU)” of AML cells using time-lapse imaging data  or day 28 (cytarabine-treated mice). Green, EGFP-expressing AML cells; red, blood vessels (Alexa Fluor 594); blue, bone tissues (SHG). Scale bar, 50 μm.Although AML chemoresistance has been reported to rely on the interaction with components of the BM microenvironment, it remains unclear whether AML cell localization within the BM can induce chemoresistance. To determine the localization of chemoresistant AML cells within the BM, we performed three-dimensional (3D) imaging using two-photon microscopy using mice injected with AML cells and subsequently treated with high-dose cytarabine at days 19 and 20 after AML cell transfer. After constructing the pseudo-surface of blood vessels and bones using the Imaris software, we calculated the distance from each AML cell to the nearest blood vessel and bone surface  software revealed that the biological functional categories “cellular movement” and “immune cell trafficking” were significantly suppressed in cytarabine-treated AML cells Fig. A, B. ThiTo further investigate the role of Rho-MRTF-SRF pathway activation in AML cell motility following cytarabine treatment, we used CCG-203971 , a CCG-1Additional file 3: Video 3. CCG treatment induces dynamic changes in chemoresistant AML cells. AML-transplanted mice were treated with CCG before/during chemotherapy and after chemotherapy, followed by intravital time-lapse BM imaging. Green, EGFP-expressing AML cells; red, blood vessels (Alexa Fluor 594); blue, bone tissues (SHG). Scale bar, 50 μm.Using intravital BM imaging, we showed that high-dose cytarabine treatment suppressed the migration of C1498 murine AML cells, consistent with a previous study using an MLL-AF9 AML mouse model . AdditioHere, we demonstrated that the Rho-MRTF-SRF signaling pathway was strongly upregulated in chemoresistant AML cells. Rho-MRTF-SRF pathway activation enhances cancer cell motility in solid tumors, leading to cancer cell migration and metastasis. MRTF-SRF pathway inhibition using CCG-1423 or its derivative CCG-203971 exerted anti-cancer effects in metastatic malignant melanoma and breast cancer , 21. OurAdditional file 4: Supplementary Figure 1. Intravital two-photon imaging at different phases of progression. Representative intravital two-photon MIP skull images from C57Bl6 mice 10 days (left), 18 days (middle), and 27 days (right) after AML cell injection (day 0). Green, EGFP-expressing AML cells; red, blood vessels (Alexa Fluor 594); blue, bone tissues (SHG). Scale bar, 50 μm. See also Video 1.Additional file 5: Supplementary Figure 2. Survival of cytarabine-treated AML-recipient mice. (A) Kaplan–Meier plot showing survival of AML-injected mice treated with PBS or 200 mg/kg of cytarabine. . **P < 0.0332 (log-rank test). (B) Scatter plots showing the cell number of AML cells included in 10,000 of BM cells. Data are presented as mean ± SD. *P < 0.05 (Kolmogorov–Smirnov test).Additional file 6: Supplementary Figure 3. Activation of SRF-MRTFA pathway after chemotherapy. (A) Top biological functions of the differentially expressed genes between chemotherapy-treated and control AML-recipient mice. (B) Heatmap showing differential expression genes after cytarabine treatment, as well as diseases and biological functions they are involved in. (C) Top five master regulators determined by causal network analysis.Additional file 7: Supplementary Figure 4. Localization of AML cells in the BM after CCG treatment.  Distribution of distance between AML cells and the bone surface (B) or blood vessels (D) after CCG treatment. Pooled data from three mice per condition from independent experiments are shown. -CCG, n = 250; +CCG, n = 130.  Mean distance between AML cells and the bone surface (B) or blood vessels (D). NS, not significant (Kolmogorov–Smirnov test).