Acute myeloid leukaemia (AML) is a rare but severe form of human cancer that results from a limited number of functionally cooperating genetic abnormalities leading to uncontrolled proliferation and impaired differentiation of hematopoietic stem and progenitor cells. Before the identification of genetic driver lesions, chemically, irradiation or viral infection-induced mouse leukaemia models provided platforms to test novel chemotherapeutics. Later, transgenic mouse models were established to test the in vivo transforming potential of newly cloned fusion genes and genetic aberrations detected in patients’ genomes. Hereby researchers constitutively or conditionally expressed the respective gene in the germline of the mouse or reconstituted the hematopoietic system of lethally irradiated mice with bone marrow virally expressing the mutation of interest. More recently, immune deficient mice have been explored to study patient-derived human AML cells in vivo. Unfortunately, although complementary to each other, none of the currently available strategies faithfully model the initiation and progression of the human disease. Nevertheless, fast advances in the fields of next generation sequencing, molecular technology and bioengineering are continuously contributing to the generation of better mouse models. Here we review the most important AML mouse models of each category, briefly describe their advantages and limitations and show how they have contributed to our understanding of the biology and to the development of novel therapies. DNMT3A, TET1, TET2, IDH1, IDH2) and chromatin modification , cellular signalling pathways of proliferation and survival , key transcriptional regulators of haematopoiesis , tumour suppressor genes , RNA splicing  and formation of cohesions complex and chromatin architecture  [DNMT3A, TET2, IDH, spliceosome mutations) that are often identified as molecular markers of potential pre-leukemic states such as clonal haematopoiesis of indeterminate potential (CHIP) and myelodysplastic syndromes (MDS) [Acute myeloid leukaemia (AML) is a disease of an uncontrolled clonal proliferation of abnormal myeloid stem and progenitor cells in the hematopoietic tissue. The transformed myeloid cells or ‘leukemic blasts’ exhibit aberrant differentiation and accumulate in the bone marrow (BM). This process diminishes normal haematopoiesis, often leading to thrombocytopenia and anaemia, hematopoietic failure and mortality . The gen, STAG2) . In factes (MDS) .Although improved modern technologies have simplified the detection of genetic alterations in AML cells, the challenge remain in validating their function during initiation and development of the disease. These alterations are categorized as either potential driver mutations necessary for disease induction and/or maintenance or neutral passenger mutations that may not be part of disease aetiology. Despite improvement in ex vivo cell culture systems, significantly expanding primary AML blasts while preserving their naïve characters over a long period remains a challenging task . In addiA large number of studies that date from the last century have shown that AML can be spontaneously triggered in mice by chemical compounds, irradiation or particular viral infections A. NotablOne of the first reported and widely used leukaemia model is the L1210 cell line (L stands for Lloyd Law) isolated from DBA/2 mice exposed to the carcinogen 3-methylcholantrene ,10. The Leukaemia was one of the first malignancies reported as a radiation-induced cancer. Leukaemia incidents were significantly higher among X-ray workers and scientists working in close proximity to particle accelerators, especially before the introduction of safety measures . SurvivoSfpi1 gene encoding for the PU.1 transcriptional master regulator of myeloid differentiation shed some light on the underlying mechanism of disease initiation [Sfpi1 (PU.1) allele is not sufficient to induce a myeloid malignancy, despite the cells having a growth advantage [Sfpi1 (PU.1) allele in its DNA binding domain (R235), is believed to transform these cells leading to clonal expansion and cancer [TEL-AML1 (aka ETV6-RUNX1) fusion, coupled with a loss of the CDKN2A cell cycle regulator gene [Radiation-induced AML (RI-AML) in the RFM mouse line correlates with human data with comparable time of exposure to leukaemia development latency ,25. The dvantage . A “secod cancer ,31. Sequd cancer . Experimtor gene . This fiMuLV) have been widely used to model the disease in susceptible mouse strains [MuLV supernatants into new-born mice [Gross-MuLV, Friend-MuLV, Moloney-MuLV, Graffi-MuLV and Rauscher-MuLV. Each of these virus strains results in recognizable and predictable patterns of disease in particularly susceptible mice strains, such as NIH/Swiss, DBA/2, AKXD, BXH-2 and C57BL-6 [SFFV) and a replication competent murine leukaemia virus (F-MuLV) [SFFV strains have been identified: (PSFFV) which reproducibly lead to polycythaemia and (ASFFV) which results in anaemia. The target cell in which both SFFV express their pathogenic effect is an erythropoietin (EPO)-responsive progenitor cell identified as a late erythroid burst forming unit (BFU-E) or colony-forming unit (CFU-E). The envelope protein encoded by SFFV interacts with and activates the EPO receptor and sf-Stk (a truncated form of the Stk/RON receptor tyrosine kinase) causing EPO-independent proliferation, differentiation and survival. In the second stage, F-MuLV integration into the Sfpi1 locus activates the myeloid transcription factor PU.1, blocking erythroid cell differentiation. Cells from diseased mice can be serially transplanted in vivo and propagated as permanent cell lines in vitro known as murine erythroleukemia (MEL) cells [Murine leukaemia viruses ( strains . Pioneerorn mice . Histori C57BL-6 . The ori(F-MuLV) ,38. Two L) cells . SubsequL) cells ,41.Akv-1 and Akv-2, and DBA/2J.) inbred mouse strain to identify putative leukaemia-inducing oncogenes through insertional mutagenesis [ecotropic virus integration site-1 (EVI1) gene on 3q26 in the human genome, today a well characterized molecular marker in aggressive AML, was identified by analysis of retroviral integration site in MuLV-infected diseased AKXD mice [Sleeping Beauty) systems [Virally induced AML was also studied in the AKXD  gene promoter [The molecular revolution in biological methods in the 70–80s of the last century allowed researchers to transfer foreign genetic elements into the germline of mice to create homogenous transgenic lines. To study the activity of putative proto-oncogenes, researchers integrated expression cassettes and mini-genes with promoter/enhancer elements, open reading frames (ORF) and transcript stabilization elements. A linearized copy of the engineered DNA is introduced by either direct injection into mouse oocytes or by electroporation into mouse embryonic stem (ES) cells B. Drivenpromoter ,49, sevePML-RARA fusion gene resulting from the t chromosomal translocation present in the vast majority of patients with acute promyelocytic leukaemia (APL). Hereby different regulatory elements directing transgene expression towards the myeloid lineage derived from human/mouse cathepsin G (CG) [CD11b [MRP8 [S100A9) genes were used. The rather wide spectrum of the resulting phenotypes illustrates the complexity and limitations of this classical transgenic approach. Whereas expression of hCG or MRP8 controlled PML-RARA expression was able to induce AML or APL-like phenotypes with incomplete penetrance after long latency [CD11b/PML-RARA transgenic mice did not develop any leukaemia [PML-RARA indeed is the genetic driver of APL and to study the underlying molecular mechanisms leading to the first (and so far only) really efficient targeted AML therapy based PML-RARA degradation by all-trans-retinoic acid (ATRA) and/or arsenic trioxide [PLZF-RARA fusion gene derived from t revealed that ATRA was unable to induce remission in mice, faithfully recapitulating the clinical response in the respective patients [The “classical” approach to establish a transgenic AML mouse model is based on direct injection of DNA fragments containing an ORF of the desired genetic and regulatory sequences into the pro-nucleus of fertilized oocytes. The zygotes are then transplanted into pseudo-pregnant foster mother mice. This results in a random integration of the transgene and founder animals are typically identified by either restriction enzyme digests and Southern blotting or genomic PCR assays . Severaln G (CG) , CD11b [) [CD11b  or MRP8 1b [MRP8  (S100A9) latency ,53, CD11eukaemia . Neverthtrioxide . Notablypatients . We listpatients ,57,58. Upatients . TherefoMLL-AF9 fusion gene associated with myelomonocytic leukaemia [MLL; aka KMT2A) gene encodes for a regulator of self-renewal and differentiation of hematopoietic stem cells (HSC) and is target of recurrent chromosomal translocations that lead to fusions of its amino-terminus to the carboxy-terminus of one of > 60 different partner loci [MLL-AF9, MLL-ENL and MLL-AF4 fusion genes, respectively [MLL-AF9 fusion from its native regulatory elements, Rabbitts and co-workers successfully integrated a short MLL exon8-AF9 cDNA-poly-A fragment into the mouse Mll1 locus by homologous recombination [Mll1 promoter, chimeric mice only developed AML. Notably, joining Mll1 exon8 with a bacterial lacZ gene was sufficient to induce leukaemia in some chimeric mice after prolonged latency [Mll-AF9 knock-in mouse line demonstrated pre- and postnatal stepwise progression of the disease [The site of integration of the cloned DNA can be specifically directed toward the desired gene locus thanks to the development of targeted homologous recombination (HR) in murine embryonic stem (ES) cells . This teeukaemia . Physiolner loci . MLL fusner loci . The mosectively . To exprbination . Interes latency . Subsequ disease , the rol disease  and gene disease .RUNX1-ETO fusion gene into the murine Runx1 promoter lead to embryonic lethality and a lack of definitive haematopoiesis in the foetal liver, very similar to those seen in −/−Runx1 knockout mice [CBFβ-MYH11 fusion gene. The resulting fusion protein was shown to interact with RUNX1 and to outcompete binding of wild-type CBFβ in a dominant-negative fashion. Not surprisingly, expression of a single copy of CBFβ-MYH11 from the Cbfb promoter (+/MYH11Cbfb) resulted in a similar lethal phenotype as −/−Runx mice [The success of modelling AML by constitutive integration of a driver fusion oncogene into its natural locus encouraged researchers to model the function of other alterations. The fusion genes of the core-binding factor (CBF), a heterodimeric essential HSC regulator composed of RUNX1 (AML1) bound to CBFβ, is involved in balanced chromosomal rearrangements found in 20–30% of human AML . RUNX1, out mice . CBF is −/− mice . Thus, tLoxP or Flp recombinase recognition target site (FRT) cleavage sites between the promoter sequence and the ORF of interest. In presence of a C- (Cre) or FLP recombinase, the STOP cassette is removed, allowing the expression of the transgene. The same approach can also be used to ablate essential parts of a gene of interest by deleting regions flanked by LoxP sites. Utilizing this setting, transgenic mice carrying the floxed genes are usually crossed with transgenic lines that express the Cre recombinase in the hematopoietic tissue. Cre expression is typically driven under the control of spatially and/or temporally controllable promoters such as the hematopoietic-restricted Vav1 (Vav1-iCre) promoter, the interferon-inducible Mx1 promoter (Mx1-iCre) or as a fusion to a mutated oestrogen receptor (ER) ligand binding domain (Cre-ER), which can be activated by tamoxifen (TAM) [Conditional gain-of-function models of AML-driving (fusion)-oncogenes are mostly generated by inserting a strong translational and transcriptional termination (STOP) sequence flanked by en (TAM) .tTA) or a reverse-tTA (rtTA) transcriptional activator respectively [RUNX1-ETO expression [RUNX1-ETO knock-in mice [RUNX1-ETO fusion transgenes in the BM upon tetracycline withdrawal in the first model and efficient excision of the floxed STOP codon in the second model, no leukaemia developed. In subsequent studies, researchers showed that mice conditionally expressing RUNX1-ETO developed leukaemia only upon treatment with genotoxic agents such as N-ethyl-N-nitrosourea (ENU) [RUNX1-ETO leukaemia model that allows for conditional and reversible controlled mosaic expression of the fusion in hematopoietic progenitors was established by transplanting whole BM carrying a ROSA26-iM2-tetO DOX inducible promoter and the fusion cDNA (ROSA26-iM2-tetOGFP/TgPtet-AML1-ETO) into lethally irradiated mice [+ AML. Transcriptional analysis from different hematopoietic populations during disease progression demonstrated that the fusion alters the transcriptional expression of HSC and committed progenitors. However, despite showing signs of a myeloproliferative leukaemia-like disease, all the mice survived. This finding is consistent with the idea that RUNX1-ETO expression is necessary but not sufficient to induce a fully penetrant AML. Indeed shRNA-targeted degradation of the fusion significantly reduced proliferation and survival of RUNX1-ETO-expression AML cells [RUNX1-ETO with mutations in tyrosine kinases such as c-KIT, FLT3-ITD or the TEL-PDGFβR fusion in different mouse models [An additional layer of complexity can be added by engineering transgenic cassettes controlled by minimal promoters that are sensitive to chemical inducers like tetracycline (Tet) , or one ectively . A Tet-opression . Another-in mice . Howeverea (ENU) . ENU is ea (ENU) . A RUNX1ted mice . Hereby ML cells . Later se models ,85.CBFβ-MYH11 fusion gene, called +/56MCbfb [Cbfb cDNA  flanked by LoxP1 sites was inserted into intron 4 of the previously generated transgenic +/MYH11Cbfb knock-in allele. Hereby the wild-type Cbfb transcript is temporarily expressed from the ‘’floxed’’ 56MCbfb allele. However, in presence of Cre, the knock-in allele is restored and a Cbfβ-MYH11 fusion is expressed. Strikingly following injection of polyinosinic:polycytidylic acid (poly(I:C)) activating Mx1-iCre, 90% of the mice developed AML after a median latency of 5 months demonstrating that the fusion is indeed a driver of AML [A similar conditional mouse model was developed for the bfb+/56M . Wild-tyr of AML .MLL-AF9, conditional expression of the MLL-ENL fusion using the same conditional Tet-on system was not able to transform GMP but induced a rather mixed myeloid-lymphoid leukaemia when activated in HSC, lymphoid-myeloid progenitor population (LMPP) or CMP [We have established a series of Tet-regulated transgenic mice to model acute leukaemia driven by the most prevalent MLL fusion genes ,66. We w) or CMP .NPM1 was regulated by the human MRP8 promoter [Npm1 [NPM1 mutant cDNA integrated in the Hprt locus followed by transplantation into irradiated WT mice induced a late-onset AML-like disease in about 30% of the recipients [NPM1c knock-in allele in the hematopoietic system (mediated by Mx1-iCre) resulted in the development of late onset AML in about 30% of the mice, however this percentage increased to 80% following the activation of cooperating proto-oncogenes through the use of the Sleeping Beauty insertional mutagenesis system [Several transgenic mouse models have been generated to model AML carrying NPM1 mutations . Conventpromoter  and a kner [Npm1  developecipients . Conditis system . CollectFlt3 slowly developed a myeloproliferative disease but no acute leukaemia [ITDFlt3 models were instrumental to demonstrate the impact of the gene dosage, loss of the wild-type allele and FLT3 ligand on phenotype development [Transgenic mouse models have also been established to model the role of the Flt3 (Fms-related tyrosine kinase 3) internal tandem repeats (FLT3-ITD) mutation found in > 20% of human AML . Two indeukaemia ,92. Howeelopment ,94.G12DK-RAS and G12DN-RAS mutations resulted a highly penetrant myeloproliferative phenotype but was not sufficient to induce AML [NPM1c with ITDFlt3 knock-in strains revealed a powerful molecular synergy with the development of highly penetrant acute leukaemia [ITDFlt3 expression was also shown to cooperate with PTDMll [NUP98-HOXD13 fusion [R394WWt1 [Dnmt3a [G12DN-Ras knock-in strain with transgenics expression of the MLL-AF9 fusion gene [BCL2 [Cbfβ-SMMHC fusion [G12DK-RAS increased the penetrance of the APL-like phenotype in cathepsin-G driven PML-RARA transgenic mice [per se not sufficient to induce the disease but act as potent cooperating lesions.Very similar to FLT3 mutations, activation of conditional transgenic knock-in alleles of AML-associated duce AML . Thus, teukaemia . Transgeh MllPTD , the NUPWt1R394W  mutation [Dnmt3a  haploinsion gene , the antne [BCL2  or the CC fusion . Transgenic mice . CollectEVI1 gene mediated by 3q21-3q26 chromosomal translocations or inversions leading a hallmark of particularly aggressive AML was recently established [Gata2 gene. To test whether this region possess enhancer activity, researchers established a transgenic mouse line with a fluorescent reporter cloned 186 kb downstream of 5’ sequences flanking the Gata2 gene [Gata2 distal hematopoietic enhancer (G2DHE). The same researchers then established a bacterial artificial chromosome (BAC) transgenic mouse that allowed the induction of EVI1 expression with or without G2DHE region. All mice with an intact G2DHE developed leukaemia in accordance with transgene copy number, where two copies gave rise to B-cell, three copies resulted in myeloid and four copies led to mixed lineage leukaemia within 200 days. However, mice lacking the G2DHE region did not show sign of disease during the 400 days of observation indicating that the GATA2 enhancer plays a critical role [GATA2 enhancer led to EVI1 silencing, growth inhibition and differentiation of human AML cells with inv(3)(q21q26) or t [EVI1 expression is under the control of a Tet inducible  promoter (TO/+/Rosa26rtTAEvi1) [TO/TOEvi1/rtTARosa26 with WT BM cells. Using this approach all mice developed symptomatic AML within 90–119 days, clearly demonstrating its oncogenic activity [A transgenic mouse line remodelling the aberrant expression of the ablished ,106. Allq21;q26) . More rea26rtTA) . To recaactivity .Gata1 gene in erythroid cells versus HSC was shown to be depended on untranslated regulatory elements located at its 5’ region [Transgenic mouse models of leukaemia have been vital for our understanding of the role of genetic aberration in the induction and maintenance of the leukemic condition. However, one of the main shortcomings of these models is their inability to reliably reproduce the leukemic phenotype observed in patients carrying the genetic lesion. Several factors could attribute for that; such as the evolutionary difference between the human and mouse haematopoiesis systems, the effect of unaccounted genetic variability in the human genome  and generating transgenic mouse lines with the cDNA sequence only. This might subsequently lead to the potential loss of essential regulatory elements located in the intronic regions of mutated genes, causing an alteration in the dynamic expression of the genetic lesion in targeted cells and thus phenotypic differences. For example, the dynamic expression of the ’ region ,110.BCR-ABL fusion gene [BCR-ABL fusion cDNA induced hematologic malignancies in about half of the recipients: either a CML-like myeloproliferative syndrome, acute lymphoblastic leukaemia (ALL) or tumours containing macrophage-like cells occurring after mean latencies of 9, 14 and 16.5 weeks respectively. Notably they were able to transfer the disease phenotype by transplanting tumour cells into irradiated secondary recipients.Technologies developed during last two decades of the 20th century allowed to transfer the cloned leukaemia-associated genetic aberrations into hematopoietic cells to explore their transforming potential in vitro and in vivo. Production of replication-incompetent high titre retrovirus expressing a gene of interest was critical to develop the widely used adoptive transfer protocol to model the effects of leukaemia-associated genetic lesions in hematopoietic stem and progenitor cells (HSPC) of the mouse. Hereby, virally transduced cells are transplanted into lethally or sub-lethally irradiated syngeneic recipients, resulting in chimeric animals in which the donor-derived transformed HSPC may outcompete the host haematopoiesis ultimately leading to leukaemia C. The poion gene . TransplCBF, RARA or NUP98 cooperating with FLT3 or N-/K-RAS mutations [BCR-ABL, AML-associated MOZ-TIF2 [MLL-AF9 [MLL-ENL [AML1-ETO [MLL-GAS7 [meningioma 1 (MN1) gene, often overexpressed in aggressive AML [Following this landmark study, this approach, often referred to as the transduction-transplantation model, was further refined and successfully used to model the transforming activity of a large number of AML-associated genetic alterations . BM recoutations . Some ofMOZ-TIF2 , MLL-AF9[MLL-AF9 ,116, MLL[MLL-ENL ,118, AMLAML1-ETO  and MLL-MLL-GAS7  fusion gsive AML .murine stem cell virus (MSCV)-based expression vectors, which allow efficient transduction and stable transgene expression in hematopoietic progenitor cells [In the majority of these studies, researchers used replication-deficient or cells . Howeveror cells ,115,116.nude (nu), severe combined immunodeficient (SCID), non-obese diabetic (NOD), NOD-SCID and NOD-SCID-IL2rγnull (NSG) strains [Ex vivo maintenance and expansion of even the most clinically aggressive patient-derived leukemic blasts remains a technical challenge. Even very sophisticated culture systems cannot fully replace the complex interactions between leukaemia cells and the BM microenvironment. To overcome these limitations researchers explored transplantation of human primary AML cells into immune compromised mice D. Severa strains ,124.nude mice that are athymic due to a homozygous nude mutation (encoding for a forkhead box transcription factor (FoxN1), resulting in lack of functional T cells. However, due to an intact B cell and NK cell function, grafting of normal as well as leukemic cells remained poor and was often associated with formation of extramedullary granulocytic tumours [nude, beige  and Xid  xenografting of human AML cells remained inconsistent and unreliable [protein kinase DNA-activated catalytic polypeptide (Prkdc) gene, which protein product is involved in DNA repair pathways. This leads to improper immunoglobulin V-D-J gene recombination, subsequently resulting in mice lacking functional mature T and B cells, however retaining NK function [+/CD38− compartment [−/−IL2Rγ mice resulted in an even more immune compromised  strain [IL2Rγ) led to an almost complete absence of the murine immune system and improved AML engraftment [hIL3, hGM-CSF and hSCF)  [W/V and NSGW41 mouse strains were obtained by breeding NSG with strains carrying c-kit loss-of-function alleles (WV/WVKit and/or W41/W41Kit) [c-Kit impairs HSCs of the host and thereby creating empty BM niches leading to a competitive advantage for transplanted human HSPC. These strains supported engraftment of human CD34+ cord blood cells (CBCs) without prior host irradiation. They also showed greater engraftment and appropriate differentiation of human cells of the erythroid and megakaryocytic lineages [In one of the first PDX experiments, researchers transplanted primary AML cells into  tumours . Even inreliable . The devfunction . Althougfunction . To overfunction . They shfunction . Moreovefunction . The abipartment ,131. Cro) strain . Deletioraftment . To furtr NSG-S) ,135. NSGr NSG-S) ,136. NSGr NSG-S) ,138. Notr NSG-S) . More reW41/W41) . Loss oflineages . In addilineages ,143,144.+ leukaemia in NSG mice resulted in a reduction in residual disease burden [Many compounds that showed significant anticancer effects in vitro and in transgenic mouse models failed to show efficacy in clinical trials, most likely due to the unaccounted complexity of the mutational load of human AML and effect of the microenvironment . To circe burden . Doxorube burden . In anote burden . Thus, tAlthough AML xenotransplantation into immunodeficient murine models is a valuable tool for the expansion and study of some aspects of the biology of human AML, these models are still limited by their inability to address the interplay of leukemic blasts with different cells of the immune system and to dissect the cell autonomous from cell non-cell autonomous aspect of the disease as they tend to develop other spontaneous malignancies. To overcome these limitations, scientists took advantage of new advances in the fields of bioengineering and synthetic material development to create biological inserts or scaffolds . The funIn an attempt to study the heterogenous sub-clones detected in human patients  and follintra and inter heterogeneity.These novel approaches in leukemic remodelling using the classical PDX system have provided proof of concept solutions on how to overcome some of challenges associated with the system, such as the difference between the mouse and human BM microenvironment and AML samples the Cas9 and a green fluorescent marker (eGFP), while the other carried the guide RNA (sgRNA) targeting the Tet2, Runx1, Dnmt3a, Nf1, Ezh2 and Smc3 genes in conjunction with another fluorescent marker (RFP-567). Viral transduction of lineage marker-depleted Sca1+; cKit+ (LSK) cells from C57B1/6 wild-type and Flt3-ITD knock-in mice with pooled sgRNA virus followed by transplantation into lethally irradiated recipients caused significant myeloid skewing of haematopoiesis and development of splenomegaly and leukaemia in some mice. Sequencing of genomic DNA from clonal leukemic cells revealed mutations in Tet2, Dnmt3a, Runx1, Nf1 and Ezh2 in single cells, thus indicating clonal outgrowth and transformation. This strategy was further refined to model mutations associated with CHIP [Dnmt3a, Ezh2, Nf1, Runx1, Ascl1, Smc3 and/or Ep300) were edited simultaneously in murine HSPC, followed by transplantation into lethally irradiated recipients [Dnmt3a mutations. The mice showed a general increase in myeloid chimerism and clonal expansion reminiscence of CHIP. Some mice died of severe anaemia, while others progressively developed hematopoietic failure and AML. Genomic DNA sequencing detected deleterious mutations in all targeted genes except Ep300, leading to block in differentiation and activation of RAS-MAPK pathway [TET2, ASXL1, DNMT3A, RUNX1, TP53, NF1, STAG2 and SMC3 in human umbilical cord blood (UCB) and adult CD34+ cells, by introducing a pool of 11-targeted sgRNAs [TET2, DNMT3, EZH2, TP53 and NF1 but only single allele mutations of SMC3, ASXL1 and RUNX1 were detected. To employ the multiplex genome editing in vivo, the researchers edited human adult CD34+ cells with the same pool of sgRNAs together with FLT3-ITD and mutated NPM. Transplantation of edited cells into immune-compromised NSG-S mice resulted in the development of CHIP and MDS. Genomic DNA screening of in vivo expanded clones showed mutagenic pattern similar to the in vitro experiments, with overrepresentation of clones carrying LOF mutation (mostly deletions leading to frame shift) in TET2, DMNT3A and ASXL1. Despite these promising observations, none of the mice developed AML. The authors suggested that differences between the human and murine BM microenvironment might impair in vivo leukemogenesis [The recent development of gene editing tools such as the clustered regularly interspaced short palindromic repeats (CRISPR) and the transcription activator-like effector nucleases (TALEN) offer novel tools to study the biology of AML by engineering disease-associated mutations in primary cells C,D. Pionith CHIP . Severalcipients . Genomic pathway . A simild sgRNAs . Consistogenesis .7q deletion associated with MDS and AML [mixed lineage leukaemia 3 (MLL3) gene but mutations and deletions of MLL3 have been only detected on one allele, suggesting that MLL3 functions as a haplo-insufficient tumour suppressor. To prove this hypothesis, researchers transduced tumour-prone (−/−p53 with reduced expression of the tumour suppressor Nf1) HSPC with sgRNA targeting Mll3 followed by transplantation into sub-lethally irradiated C57Bl/6 mice. Compared to controls, targeting Mll3 significantly accelerated leukaemia development. Subsequent gDNA sequencing of individual Mll3 edited AML clones revealed both wild type and mutant alleles in the majority of samples. This suggested that leukemogenesis selects for partial but not complete Mll3 inactivation, providing compelling evidence that Mll3 is a haplo-insufficient tumour suppressor gene in AML [Another study used CRISPR/Cas9-based genome editing to model the recurrent  and AML . The come in AML .MLL and AF9 genes (MLL-AF9 and AF9-MLL) in primary human CD34+ UCB-derived HSC, to recapitulate MLL rearrangements in patients’ cells [+ UCB cell [Genome editing using TALEN effector nucleases was used to specifically generate reciprocal chromosomal translocations of the s’ cells ,160. In s’ cells . In contUCB cell . UnfortuNone of the currently used mouse AML models faithfully recapitulate the complex biology, cell to microenvironment interactions and dynamic progression of AML. Nevertheless, they have been instrumental in deciphering the underlying pathology of the disease and advancing AML research. Historically, the chemical, irradiation and viral models set the field of AML modelling in mice and were used to develop many AML drugs. Transgenic mouse lines harbouring AML associated mutations have enabled researchers to directly link genetic aberrations to AML initiation and progression. The creation of immunocompromised mouse strain has allowed for the expansion and study of human primary AML cells and the discovery of a hierarchy led by leukemic stem cell. In the future, advancement in genome editing technologies and collaboration between multidisciplinary fields would lead to the generation of more humanized mouse strains, which will ultimately help scientists to accurately model the complex biology of AML in mice.


To fully understand the role of calcium redistribution upon excitation and contraction in skeletal muscle cells, the local [Ca2+] in different compartments needs to be taken into consideration. Fluorescent probes allow the determination of [Ca2+] in the cytosol where myofibrils are embedded, the lumen of the sarcoplasmic reticulum (SR) and the mitochondrial matrix. Previously, models have been developed describing intracellular calcium handling in skeletal and cardiac muscle cells. However, a comprehensive model describing the kinetics of the changes in free calcium concentration in these three compartments is lacking. We designed a new 3D compartmental model of the half sarcomere with radial symmetry, which accounts for diffusion of Ca2+ into the three compartments and simulates its dynamics at rest and at various rates of stimulation in mice skeletal muscle fibers. This model satisfactorily reproduces both the amplitude and time course of the variations of [Ca2+] in the three compartments in mouse fast fibers. As an illustration of the applicability of the model, we investigated the effects of Calsequestrin (CSQ) ablation. CSQ is the main Ca2+ buffer in the SR, localized in close proximity of its calcium release sites and near to the mitochondria. CSQ knock-out mice muscles still preserve a near-normal contractile behavior, but it is unclear whether this is caused by additional SR calcium buffering or a significant contribution of calcium entry from extracellular space, via stored-operated calcium entry (SOCE). The model enabled quantitative assessment of these two scenarios by comparison to measurements of local calcium in the cytosol, the SR and the mitochondria. In conclusion, the model represents a useful tool to investigate the impact of protein ablation and of pharmacological interventions on intracellular calcium dynamics in mice skeletal muscle.Variations of free calcium concentration ([Ca When the RyR channels are closed (low RyR permeability), [Ca2+]cyto drops below the threshold required to activate the actin filaments and the muscle relaxes. Ca2+ uptake in the mitochondria plays an important role in modulating the resynthesis of ATP required for force generation and SR Ca2+ reuptake via SERCA.In skeletal and cardiac muscle cells, calcium ions are stored in the TC of the SR which are in close contact with the TT system, which propagates the action potential (AP) during excitation-contraction (EC) coupling. The AP triggers the release of calcium through the interaction between DHPR and RyR, so that calcium can diffuse in the cytosol. The binding of calcium to the Tn-C subunit of troponin induces a shift of the tropomyosin filament on the actin filament uncovering the actin-binding sites for myosin and allowing for the beginning of the acto-myosin cycle and force generation with the associated chemical energy (ATP) utilization. The release of calcium via RyR channels, its binding to cytosolic buffers, and the re-uptake back into SR through the sarcoplasmic endoplasmic reticulum calcium ATPases (SERCA) determines the amplitude and the time course of the cytosolic calcium  and the mitochondrial matrix, can be achieved by more recently developed genetically targeted FRET-based fluorescent probes  during contractile activity in the three compartments. As a further test and an illustration of the applicability of the model we investigated the effects of Calsequestrin (CSQ) ablation. In our laboratories, wild type mice and mice carrying null mutations of the genes coding for calsequestrin (CSQ-KO) have been comparatively studied for several years cyto in the 8th longitudinal and 4th radial sub-compartment, 125 nm away from RyR. The distribution of these elements has been chosen to closely match the observed average position of mitochondria at about 130 nm distance from the TC [2+ signal in frog skeletal muscle from an 18- and a 100-compartments model have been reported to be close each other [2+] increases by 9% (from 870 to 950 nM), while the minimum is almost stable at 240 nM  increases and decreases exponentially with time constants τon = 1 ms and τoff = 5 ms. Then, the rate of Ca2+ release is:max is the maximum permeability which includes the release surface of the TC. The rate of release depends on the gradient of [Ca2+] between the TC and the sub-compartment of EF adjacent to TC (subscript x). Actually, the rate of calcium release depends on several factors which have been collected in the parameter "evacuability" [max to obtain the experimentally observed averaged [Ca2+]cyto in WT mice during the steady phase of the train of stimuli. This approach does not take calcium dependent inactivation into account. A reduction in the Ca2+ release flux from the first AP to the second and subsequent has been considered in previous models [2+] in the three compartments during the steady phase, reached toward the end of a stimulation train. Therefore, we decided not to include such a phenomenon. The impact of calcium dependent inactivation, as described previously (27), in the present model is illustrated and discussed in the Caposed in , we assuability" ,28, whicability"  we fit ts models  to accoumax = 4 mmol s-1 per liter of fiber volume and a Ca2+ concentration at which half-maximum pump rate is reached, Km = 0.5 μM [2+]cyto in quiescent cells constant.SERCA activity is modeled by simplified first-order kinetics proposed by , as foll= 0.5 μM . This im2+ transients. Our model validation is based on experimental data of free calcium concentration in SR and mitochondria, i.e. [Ca2+]SR, and [Ca2+]mito, respectively. In this comprehensive analysis, the diffusional approach which takes into account microdomains in the space adjacent to mitocondria or SR, becomes very important. This feature is absent in compartmental models which only consider averaged concentration within compartments.This compartmental diffusional model extends the model proposed by Cannel and Allen  by inclu2+ concentration in the mitochondrion [Ca2+]mito,tot is:MCU is the influx via the mitochondrial Ca2+ uniporter (MCU) and JNCE is the efflux through mitochondrial Na+/Ca2+ exchanger (NCE). A detailed description of fluxes of Ca2+ ions through MCU, and NCE would require the analysis of all the components which influence the mitochondria membrane potential ΔΨm which in turn affects MCU influx [+], [Na+], [K+], their flux through the inner mitochondrial membrane and relative buffers in the matrix, and would introduce several additional parameters to the model. We consider such a detailed description beyond the scope of the present work, and adopt a simpler model based on a constant [Na+] and a constant ΔΨm, which has proven to be suitable for cardiac myocytes, at relatively low frequency of stimulations [MCU is simplified as a saturable first-order transporter independent of the internal [Ca2+]mito:MCU is the maximum flux rate, [Ca2+]x the Ca2+ concentration in the sub-compartments of EF where the mitochondrion resides and Kd the Ca2+ concentration where the pump rate is half-maximal. fMCU is a multiplication factor to fit the data, h the Hill parameter and Joffset the constant flux which equilibrates the basal flux via NCE, JNCE.The study of calcium dynamics in the mitochondrion is a relatively new subject ,24,32–34U influx . This woulations . In thisNCE is based on a 3:1 stoichiometry for Na+ and Ca2+  as well as cytosolic and intra-mitochondrial [Na+]. This relationship leads to symmetrical uptake and decay transients in [Ca2+]mito. However, experimental data show that [Ca2+]mito exhibits an average rise time during train of stimuli of 27.8 ms, much faster than its exponential decay after the end of stimulation (rate constant of 250 ms-1). To account for this asymmetry, we propose a phenomenological modification of JNCE, which follows the original description proposed in [2+]mito = 1 μM, and increases exponentially above this value  in the TC, an additional buffer (AB) in the SR, Parvalbumin (PVA) in cytosolic space (both EF and MS), Troponin C (Tn-C) in MS, and a buffer in the mitochondrial matrix (B). PVA-sites can be occupied also by Mg2+ whose concentration is assumed to be constant. Values for buffering capacity and binding and unbinding rates (reported in 2+ and Mg2+), and cyto = 0.38 μM considered in this study, this event has a characteristic time of about τB = 30 ms, one order of magnitude higher than τD. We therefore introduced the simplification that, in each time step, the equilibrium between each buffer and the free calcium concentration is governed by the mean value of [Ca2+] in the cytosolic space <[Ca2+]cyto> , and  for Tn-C2+), and . The cytsee also ). In thi2+ per mole of CSQ1  in the right-hand-side. This introduces a reasonable approximation  signals, i.e. variations in free calcium concentrations, adopted as reference for fitting, and simulation were derived from data previously published, using different ratiometric calcium probes in cytosol  or R-[Ca2+] respectively) has been derived in 2+] signa cytosol . Single ameleons ) directerived in  and 14]2+] signarived in  stronglymax in 2+]cyto recorded during a stimulation train at 60 Hz; the multiplicative parameters fMCU and fNCE and the parameters in JNCE were adjusted to match the amplitude and time course of [Ca2+]mito at 60 Hz . It can be seen that the local [Ca2+] is more than two-fold higher than the average [Ca2+]cyto. This is in agreement with the concept of micro-domain [The inset in the upper right panel of o-domain  and stre2+] values in all three compartments are in fair agreement with the experimental data at all stimulation rates. Simulated traces for [Ca2+] transients in the three compartments  at 1, 5 and 20 Hz stimulation and the analysis of the kinetics are reported in The model with the parameters derived at 60 Hz stimulation was validated by considering the predictions of free calcium variations during stimulation at lower frequencies . In max, which was doubled to account for the increased number of RyRs, reported in [2+] is not as high as expected since the force generated is at least sufficient for normal mobility of sedentary mice.Next, given the long experience of our laboratories with mice carrying a null mutation of CSQ ,13,45 weorted in . Calciumorted in , thus CSorted in , see Taborted in . However2+] in all three compartments due to ablation of CSQ (data not shown). Results of the model with an additional buffer (AB) with a buffer capacity ratio relative to CSQ of 0.1 are reported in 2+]cyto has an initial peak similar to that predicted for WT, but it rapidly decreases to a lower steady value, while the [Ca2+]SR shows a much stronger depletion compared to WT. Qualitatively, these results resemble the behavior of free [Ca2+] in SR and cytosol in CSQ-KO cells [2+]mito is different from what is observed experimentally as [Ca2+]mito is not able to maintain the relatively high values observed in CSQ-KO, especially at high stimulation frequencies.The mechanisms behind these near-normal contractions are not well understood. The presence of other calcium buffering proteins in the SR, such as sarcalumenin, calreticulin, calnexin, junctate and others ,46–49, cKO cells . Quantit2+]mito in CSQ-KO, we considered that doubling of the number of RyRs might lead to an increased RyR leakage in basal conditions. Since the basal [Ca2+]cyto is the same in CSQ-KO and WT fibers, SERCA needs to pump twice as much. This will lead to a constant average [Ca2+]cyto, but higher local concentrations near the RyR. In principle, the higher local [Ca2+]cyto might allow for the observed higher basal [Ca2+]mito (230 nM in CSQ-KO vs 160 nM in WT [2+]mito by less than 1%.In an attempt to improve the prediction of cyto at 60 Hz is close to the experimental data in CSQ-KO (see 2+]SR) during 60 Hz stimulation would be lower than those observed experimentally , indicating that even in this extreme case the effect of the AB is insufficient to sustain the near-normal contraction at high frequencies. Thus, the model suggests that an extra source of external calcium (possibly via SOCE) is needed as hypothesized in SR would be influenced by the SR buffer capacity and external Ca2+-influx via SOCE. As a consequence, the total Ca2+ within the fibers would increase and the calcium entry during one contractile cycle should benefit the subsequent one, as extrusion of the Ca2+ from the fibers is expected to occur more slowly than entry via SOCE.The results showed that even the maximal buffer capacity of a secondary buffer (AB) accounting for 20% of the buffering power of CSQ in WT fibers  was insumyotubes . A recenmyotubes  suggests2+]SR. Such an amount is well below what is required to keep cytosolic levels comparable not only to those of WT muscle fibers (approximately 180 μM would be needed in addition) but also to those experimentally observed in CSQ muscles (a contribution in the order of 100 μM would be required). This latter value corresponds (within 2 s) with a flux of 50 μM/s which is, in view of the uncertainties in the experimental determinations and in the calculations, in fair agreement with the experimentally estimated value of SOCE of 19 μM/s in mito. These aspects warrant further study because quantitative data on mitochondria in living skeletal muscle fibers are still scarce.The simulation of the calcium uptake via MCU and release via NCE could be improved by taking the influence of NaIn conclusion, the present model is implemented on a robust background dating back 30 years ago ,6, but rS1 Table(PDF)Click here for additional data file.S2 Table2+] in the three compartments  using these modified parameters ([Ca2+]±5%) relative to the values obtained with the parameter values in 2+]basic) are calculated and expressed as a percentage (-5%/+5%). All percentages are lower than 5% except for the Hill parameter  in the calsequestrin equilibrium equation. The values of To evaluate the sensitivity of the model predictions on the actual model parameters, a change of ±5% has been applied to each parameter reported in (PDF)Click here for additional data file.S1 Fig2+] in the cytosol where NCE and MCU are supposed to be located (125 nm away from the RyR) and in the mitochondrion using the 50-compartments model (red) and a 300-compartments model (blue).Simulation of the mito behavior for different choices of the buffering capacity and kinetics, keeping a ratio for free-to-bound [Ca2+] of 1:100.The nature of the mitochondrial buffer is largely unknown at present, and experimental data needed for its mathematical characterization are not available in literature. We have analyzed the [Ca(PDF)Click here for additional data file.S5 File2+] in the three compartments.Effect of the kinetic term on the [Ca(PDF)Click here for additional data file.S6 File2+] for the three compartments at different stimulation rates in WT fibers and CSQ-KO fibers with 10% and 20% of total calcium bound accounted by secondary buffer LB.Time course of the variations in free [Ca(PDF)Click here for additional data file.S1 VideoThe peak corresponds to the position of the ryanodine receptors.(AVI)Click here for additional data file.


Upregulation was abolished for each of these by inhibitors of transient receptor potential channel 1 and the inositol trisphosphate receptor. L-channel [Ca2+]i inhibitors and RyR antagonists had no such effect. These results demonstrate that [Ca2+]i-dependent glycogenolysis participates in acquired glioma TMZ-resistance by upregulating MGMT via a Na,K-ATPase/ERK1/2 signaling pathway. GPBB and glycogenolysis may therefore represent novel therapeutic targets for overcoming TMZ-resistant gliomas.Understanding the mechanistic basis for temozolomide (TMZ)-induced glioma resistance is an important obstacle in developing an effective form of chemotherapy for this type of tumor. Glycogenolysis is known to play an essential role in cellular proliferation and potassium homeostasis and involves the glycogen phosphorylase isoenzyme BB (GPBB). In this investigation, plasma GPBB was correlated with TMZ-resistance. Elevated plasma GPBB concentrations were found to be more frequent in a TMZ-resistant cohort of patients with poor survival rates. TMZ inhibits cell proliferation and induces TMZ resistance by upregulating the expression of O(6)-methylguanine-DNA methyltransferase (MGMT). This process requires glycogenolysis, which was confirmed herein by treatment with 1,4-dideoxy-1,4-imino-D-arabinitol hydrochloride, a glycogenolysis inhibitor and a special GPBB inhibitor. Acute TMZ treatment leads to upregulation of [Ca Gliomas are the most common and aggressive tumors of the central nervous system. The most aggressive forms of glioma (grade III and grade IV), based on the World Health Organization’s (WHO) classification system, have limited therapeutic options with poor survival rates . Despite2+]i-dependent, not only in muscle, but also in brain slices  for [Ca2+]i release . The present study is based on a discussion by Professor Hertz that glioma GPBB upregulation, in response to TMZ, may require glycogenolysis and its associated pathways. It is important to note that roles for GPBB and glycogenolysis in glioma and TMZ resistance remain unclear.2+]i release and cell proliferation in the presence and absence of inhibitors of potential signaling pathways, with and without stimulation (cAMP or TMZ). Chronic TMZ treatment was examined for its effect on MGMT as well as an analysis of the effects of canrenone, a specific inhibitor of Na,K-ATPase that is upstream of mitogen-activated protein kinase (MAPK) and increases [Ca2+]i  butadiene] were purchased from Calbiochem . Fura-2 AM was obtained from Invitrogen .Chemicals used in the preparation of media, and most other chemicals, including DAB , cAMP (cyclic adenosine monophosphate), temozolomide, xestospongin C -eicocahydro-5H,17H-1,23:11,13-diethano-2H,4H-, dioxacycloeicosino  dipyridine, nifedipine, and canrenone were purchased from Sigma . TRPC1 channel antibody (SC-15055) was obtained from Santa Cruz Biotechnology . OligofectamineThis study was approved by the Medical Ethical Committee of Liaoning Cancer Hospital and Institute (No. 20150309-2). Informed consent was obtained from all patients and controls before enrollment in this study. All patients underwent surgery and subsequent TMZ chemotherapy, and all patients underwent a minimum of 3 years of follow-up. The diagnosis was verified by pathological analysis and classified according to the WHO’s standards.2 as described in previous study . Cell cultures were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) and cultured at 37°C in a humidified atmosphere containing 5% COus study .g for 15 min at 4°C. Plasma samples were then kept at -80°C until assayed. Plasma GPBB concentration was measured with GPBB ELISA Kits , according to the manufacturer’s instructions. Readings were made at 450 nm excitation and 570 nm emission with a microplate reader . A standard curve was used to assess intensity at different GPBB concentrations from which GPBB concentration was calculated.Blood samples, collected in EDTA tubes, were centrifuged at 1,000 ×(PCM)-2 medium  for 7 days. Neutral red was then added to stain colonies in collagen gel droplets, which were then fixed with formalin. Images of the stained gels were acquired with a video microscope  and cell proliferation rates were determined by measuring optical densities. In vitro chemosensitivity was expressed as a ratio of the total colony volume of the treated group (T) to that of the control group (C) (T/C ratio). A T/C ratio of ≤ 60% was regarded as TMZ sensitive, with TMZ resistance defined as a T/C ratio of > 60%.The CD-DST was used to evaluate the sensitivity of cancer tissue to temozolomide (TMZ) . In briePyro-sequencing was performed as previously described . BrieflyThe MGMT methylation was detected using methylation-specific PCR (MSP), as described in previous study . Genomicin situ hybridization (FISH) was performed for 1p/19q co-deletion analysis. Control and detection probes were developed from plasmids D1Z1 (1q12) and D1Z2 (1p36.3) for the chromosome 1 study and from bacterial artificial chromosomes (BACs) RP11-413 M18 (19q13) and CTZ-2571 L23 (19q13.3) for the chromosome 19 study. Dual-colored probes against chromosomes 1p and 19q were used to detect chromosomal loss at these loci. A single fluorescent signal in the nucleus was interpreted as chromosomal-arm loss if, in addition, two signals were detected for the control probe.Fluorescence Glycogenolysis determination protocols have been described previously . Briefly3 cells/well in growth medium. Cell growth was then assayed by the addition of 20 μL of MTT  to each well, and the plate was incubated at 37°C for 4 h. The proliferation assay was performed for 3 days, and cell growth was assayed every 24 h. The reaction was stopped by the addition of 200 μL dimethyl sulfoxide (Sigma–Aldrich). Optical density was measured at 490 nm with a microplate reader . The experiments were performed from three to five individual cultures of each treatment.The proliferation potential of U251 glioma cells was determined by MTT assay . Briefly, cells were seeded into 96-well plates  at a cell density of 1 × 102+]i determination protocols have been described previously  was used to record the fluorescence intensity of fura-2 AM in saline solution  for 30 min at 37°C for glioma cells grown on coverslips coated with polylysine. After two washes with this saline solution, coverslips were either perfused in the saline solution (control), or TMZ was carefully added to the saline at time zero. Readings were taken at 340 and 380 nm excitation and 510 nm emission at 20-s intervals. The incubation continued for 22 min. Between 13 and 20 cells were selected for each coverslip, and three to five coverslips were assessed in each experimental group. The represents of this experiment performed means from 38 to 81 cells on three to five individual coverslips of each treatment.The i)-mediated signaling pathways, the effect of TMZ on [Ca2+]i was assessed. [Ca2+]i (as indicated by 340/380 nm fluorescence ratio) was significantly increased by the addition of cAMP, with the increased [Ca2+]i rapidly returning to resting values over a period of 15–20 min . As shown in Figure 3B, TMZ had a similar effect on [Ca2+]i, but the release of [Ca2+]i was less than with cAMP. Under control conditions, DAB had no effect on [Ca2+]i induction by TMZ or cAMP . Overall, [Ca2+]i was not influenced by the glycogenolysis inhibitor DAB. These results suggest that glycogenolysis occurs downstream of the TMZ/cAMP-induced increase in [Ca2+]i.To identify whether glycogenolysis is inhibited by the blockade of intracellular calcium . As shown in Figure 3C, inhibition by a neutralizing antibody of the transient receptor potential channel 1 (TRPC1), an ER-activated calcium channel in the TRPC1 plus TMZ group, resulted in a significant reduction in [Ca2+]i release by TMZ. There was no difference between control cells and cells treated with TRPC1 neutralizing antibody plus TMZ. However, when U251 cells were administered with nifedipine , a dihydropyridine inhibitor of [Ca2+] L-channels, depolarization led to an increase in free cytosolic [Ca2+] concentration. [Ca2+]i was significantly higher than under control conditions and appears to be similar to TMZ alone. Similar analysis was conducted with ER receptors. The TMZ induced [Ca2+]i increase was completely abolished by xestospongin C, an antagonist of inositol trisphosphate receptor (IP3R). Ryanodine, an RyR-specific antagonist, did not significantly influence TMZ-induced [Ca2+]i release .The mechanism underlying the TMZ stimulation of glycogenolysis was assessed by analysis of the effect of i, the TMZ inhibition of cell proliferation was incompletely affected by nifedipine and ryanodine. These [Ca2+]i and cell proliferation data suggest roles for TRPC1 and IP3R, although it is not clear what the precise roles of these molecules in TMZ-induced glycogenolysis might be.The TMZ inhibition of cell proliferation was completely abolished by TRPC1 neutralizing antibody and xestospongin C, an antagonist of IP3R , xestospongin C (an IP3 receptor blocker), ryanodine (a RyR-specific antagonist), and canrenone . AKT phosphorylation was also assessed. Figures 6–8 demonstrate that ERK1/2 phosphorylation was induced by TMZ and significantly inhibited by TRPC1 neutralizing antibody, xestospongin C, and canrenone, which on their own had no effect. Compared to TMZ-treated cells, no significant change in ERK1/2 phosphorylation was observed in cells treated with nifedipine or ryanodine . However, ERK1/2 phosphorylation was increased to a higher level when compared to control cell cultures. These data suggest that TMZ-induced ERK1/2 phosphorylation requires glycogenolysis and TRPC1/IP3R-induced [Ca2+]i release.To further investigate the underlying mechanisms of TMZ-induced ERKFigure 9, chronic treatment with TMZ for 48 h resulted in a significant increase  in the expression of Na,K-ATPase, ERK1/2, and MGMT proteins, normalized to the housekeeping protein β-actin. Pretreatment with DAB, a specific antagonist of GPBB and glycogenolysis, eliminated the TMZ effect on Na,K-ATPase, ERK1/2, and MGMT proteins.The effect of chronic TMZ on the MAPK signaling pathway, and on the expression of MGMT, a crucial target for TMZ in glioma cells, was explored. As shown in Figure 10, the effect of TMZ chronic treatment on Na,K-ATPase, ERK1/2, and MGMT protein was abolished by TRPC1 neutralizing antibody and xestospongin C, an antagonist of IP3R. Figure 11 demonstrates TMZ-mediated upregulation of Na,K-ATPase expression, which was confirmed by the inhibitory effect of canrenone, a specific antagonist of Na,K-ATPase, which was similar to the effect on ERK1/2 and MGMT expression. Furthermore, the upregulation of TMZ-induced ERK1/2 and MGMT expression was abolished by U0126, an inhibitor of ERK1/2, phosphorylation, with no effect on Na,K-ATPase expression, as shown in Figure 12. Taken together, these data indicate that the inhibition of downstream signaling did not inhibit upstream signaling. Furthermore, canrenone inhibited the activation of Na,K-ATPase before the transactivation of ERK1/2 and upregulation of MGMT. Overall, as shown in Figure 13, TMZ required [Ca2+]i-dependent glycogenolysis, which led to Na,K-ATPase/ERK1/2 phosphorylation and the upregulation of these proteins, as well as MGMT activation which induced TMZ-resistance in glioma cells.As shown in 2+]i-dependent glycogenolysis is required for TMZ resistance, stimulated by both the transactivation of Na,K-ATPase/ERK1/2 and the upregulation of MGMT expression in glioma cells. This is graphically illustrated in Figure 13, wherein TRPC1 and IP3R release [Ca2+]i with activation of Na,K-ATPase and ERK1/2, the latter process was glycogenolysis-dependent and may upregulate the expression of MGMT protein.This study reports, for the first time, that plasma GPBB concentrations differ between high-grade glioma patients with TMZ sensitivity and TMZ resistance. Our results demonstrate that i concentration. The absolute [Ca2+]i dependence of glycogenolysis was first demonstrated in muscle , TRPC1 neutralizing antibody, and [Ca2+] entry, suggesting the participation of a pathway initiated by endogenous ouabains. This hypothesis was verified by canrenone inhibition (2+] influx through TRPC1 and by activation of IP3R in the ER. Recent studies have also shown that SOC operation stimulates glycogenolysis (Glycogen phosphorylation by GPBB shows an essentially identical requirement for an increase in free i release and acquired alteration of MGMT expression by glycogenolysis. Finally, the predictive value of plasma GPBB concentrations needs to be verified in a larger cohort. In addition, short follow-up times for this study may result in survival data that are inaccurate.There are some limitations to this study. First, blood samples from high-grade glioma patients were not available during treatment and hence could not be evaluated. Second, the predictive value during TMZ treatment could not be determined. Another limitation is that the mechanism underlying the activation of Na,K-ATPase and ERK2+]i-dependent glycogenolysis via the Na,K-ATPase/ERK1/2 signaling pathway.This study has shown that plasma GPBB concentration is a novel biomarker that can be used to predict the response to TMZ in high-grade glioma patients. These findings suggest that TMZ inhibits cell proliferation and induces acquired TMZ resistance by upregulating MGMT. The latter process requires [CaJX performed the literature database search and wrote the manuscript. JX and YZ performed the experiments. YZ, XG, and TS contributed to the vital insights and proofread the manuscript. JX and TS conceptualized the research topic.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Adrenal myelolipomas (AMLs) are rare benign adrenal tumors, containing adiposeand hematopoietic tissue, a result of reticuloendothelial cell metaplasia.Incidence on autopsy has been reported from 0.08% to 0.4%. AMLs are generallyconsidered nonsecretory. The functional aspect of adrenal incidentaloma shouldbe evaluated. In this article, we report a case of a 40-year-old male, whopresented with uncontrolled hypertension and renal failure, with imagingrevealing an adrenal incidentaloma. He was started on dialysis for acute fluidoverload, and workup for pheochromocytoma revealed an elevated serumnorepinephrine level of 1181 pg/mL. Free metanephrine and normetanephrine levelswere low when checked pre- and post-dialysis. Complete resection of theencapsulated right adrenal mass was performed. Pathology of the adrenal tumordemonstrates an 11.5 × 9.5 × 7.5 cm well-circumscribed, partially encapsulatedproliferation of mature adipose tissue with admixed hemopoietic elementsconsistent with myelolipoma weighing 29.3 g. This case highlights the inclusionof a full metabolic workup for all adrenal incidentalomas, including AML. The functional aspect of AI should beevaluated.Adrenal myelolipomas (AMLs) are rare benign adrenal tumors, containing adipose andhematopoietic tissue, a result of reticuloendothelial cell metaplasia. It issporadic with an overall incidence reported 0.08% to 0.4% at autopsy previously,compared with increased incidence of 10% to 15% recently due to the widespread useof imaging.A 40-year-old male was evaluated with the chief complaint of a 1-week history oftesticular and bilateral lower extremity swelling. He was found to be in ahypertensive emergency with a blood pressure of 234/119 mm Hg and needed intravenousantihypertensive medications to control the blood pressure. Other vital signs onpresentation were a temperature of 98.4°F, pulse rate of 100 beats per minute, andoxygen saturation of 92% on room air. Physical examination was significant fordecreased air entry on bilateral lung bases, bilateral lower extremity pittingedema, and scrotal edema. Past medical history was significant for hypertension,morbid obesity, chronic kidney disease stage 4, and right adrenal mass, which wasdiagnosed 5 years prior to the presentation . The patient has been prescribed multipleantihypertensives but was noncompliant.Laboratory data showed hemoglobin 8.7 mg/dL, blood urea nitrogen 70 mg/dL, andcreatinine 8.7 mg/dL, with glomerular filtration rate 7 mL/min. A CT abdomen/pelviswithout contrast revealed an interval increase in the size of hypoattenuating rightadrenal mass measuring 9.7 × 7.7 × 6.1 cm compared with 8.2 × 6.9 × 6.4 cm 6 monthsprior . OtheriThe patient was transferred to the tertiary care center due to the unavailability ofthe endocrine surgeon. At the tertiary care center, CT of abdomen and pelvis withcontrast revealed heterogeneously enhancing round adrenal lesion on the rightmeasuring 9.9 × 8.7 cm. Average attenuation is approximately 0 Hounsfield units(HU), and lesion enhances to 16 HU on venous and delayed-phase imaging, which isless consistent with pheochromocytoma. There was also little difference in the pre-and post-dialysis levels of metanephrines, which questioned the diagnosis ofpheochromocytoma. Given the history of progressively growing adrenal mass, thedecision was made to proceed with adrenalectomy. The patient underwent open rightadrenalectomy and tolerated the procedure well with no intraoperative hemodynamicinstability. A completely encapsulated adrenal tumor was removed intact. Pathologyof the adrenal tumor demonstrated an 11.5 × 9.5 × 7.5 cm well-circumscribed,partially encapsulated proliferation of mature adipose tissue with admixedhemopoietic elements consistent with myelolipoma weighing 29.3 g . The pat2 The tumor is composed of mature adipose tissue admixture with hematopoieticelements. AML involving extra-adrenal locations like thorax, liver, spleen, stomach,mesentery, pelvis, and retroperitoneum have been reported.3 Rarely they can be functional or coexist with other endocrine disorders likeCushing syndrome, congenital adrenal hyperplasia (CAH), primary aldosteronism, andpheochromocytoma. CAH has been increasingly reported with AML.4 Chronic ACTH over stimulation of adrenals may have a role in patients withbilateral AML and also in patients with untreated CAH with AML.5Adrenal myelolipoma is a benign tumor originating from the adrenal cortex and isusually nonfunctional. Gierke initially described myelolipoma in 1905, and Oberlingcoined the term “myelolipomatoses” in 1929.6 AML has been reported with thalassemia rarely and tend to be giant andbilateral, due to increased production of erythropoietin.7The comorbidities like hypertension, obesity, diabetes, atherosclerosis, andmalignancy have been associated with AML.8 AML is a clonaltumor based nonrandom X-chromosome inactivation identified on recent cytogenetic studies.7The origin of AML is unclear; few postulated mechanisms are extramedullaryhematopoiesis, hamartosis, embolism of bone marrow element, metaplasia ofreticuloendothelial cells of blood capillaries  in adrenal glands, and degeneration of adrenal corticalcells.9 Boudreaux et al described the largest AML in a patient with CAH, size 34 × 24× 10.5 cm, weight 5900 g.10Adrenal myelolipoma is usually asymptomatic and generally has a diameter <5 cm,but can vary from <1 cm to >30 cm. Sometimes the diameter of AML is >10 cm,often described as giant AML. Akamatsu et al described the largest AML without theendocrine disorder, measuring 31 × 24.5 × 11.5 cm and weighing 6000 g.11 The sensitivity and specificity of plasma catecholamines are very low at78.6% and 70.7%, respectively.12 The plasma-free metanephrines or urinary fractionated metanephrines are moresensitive and specific in pheochromocytoma.13 The CT scan also showed low Hounsfield units, which are less likely,consistent with pheochromocytoma.14The serum catecholamine levels are not reliable for the diagnosis of pheochromocytoma.15 The previous AACE guidelines published in 2009 did not recommend metabolicworkup for AML.16 Endocrine dysfunction of AML is underappreciated as the review of theliterature reveals that 7% in AML and 11% in AI were functional.8 AML should not always be excluded from metabolic workup. Endocrine workup isbeneficial in AML patient with hypertension, younger patients, diabetes orprediabetes, and those with bilateral AML.8 Function AML resection resulted in resolution of hypertension and sequelae inseveral reports, underestimating the incidence of hormonal abnormality.Recent American Association of Clinical Endocrinologists (AACE) disease stateclinical review suggested that incidentally discovered adrenal masses requiredynamic and static hormonal measurements.4 CT is more sensitive for detection than other imaging modalities.7 Ultrasonography appearance can be hyperechoic or hypoechoic depending on thepredominance of fat or myeloid cells. Similarly, CT can have a high attenuation withmyeloid tissue and low attenuation with a fatty tumor. Magnetic resonance imagingappearance of the tumor demonstrates the high signal intensity and reduced signalintensity depending on the T1-weighted or T2-weighted sequences, respectively.Retroperitoneal fat–containing tumors like teratoma, lipoma, myolipoma,angiomyolipoma, and liposarcoma may mimic AML radiologically.8Adrenal myelolipoma can be diagnosed in 90% of the cases by ultrasonography, CT, andmagnetic resonance imaging.17Adrenal biopsy should be considered in the following circumstances as per endocrineliterature: (1) hormonal inactivity of the adrenal mass, especially ruling outpheochromocytoma; (2) benign characteristics of an adrenal mass not established onimaging; and (3) biopsy results of adrenal mass would alter the management.4 Surgical removal is indicated if the patient is symptomatic, tumor >5 cm,increased risk of rupture, or if malignancy is suspected. Spontaneousretroperitoneal hemorrhage is a well-recognized complication of AML, which is rare.4Pathological diagnosis of AML requires the presence of hematopoietic elements andmature adipocytes. Management of AML is dependent on the size of the tumor. Tumors<5 cm are generally asymptomatic and can be monitored intermittently with imaging(1-2 years).This case highlights the inclusion of a full metabolic workup for all adrenalincidentalomas, including AML. The serum catecholamine levels are not reliable fordiagnosis of pheochromocytoma, but measured plasma metanephrine levels or urinaryfractionated metanephrines are more sensitive and specific in pheochromocytoma.


The tubular system is the invagination of the plasma membrane that forms junctions with the sarcoplasmic reticulum (SR) where STIM1, Orai1 and ryanodine receptors are found. The physiological activation of SOCE in muscle is not defined, thus clouding its physiological role. Here we show that the magnitude of a phasic tubular system Ca2+ influx is dependent on SR Ca2+ depletion magnitude, and define this as SOCE. Consistent with SOCE, the influx was resistant to nifedipine and BayK8644, and silenced by inhibition of SR Ca2+ release during excitation. The SOCE transient was shaped by action potential frequency and SR Ca2+ pump activity. Our results show that SOCE in skeletal muscle acts as an immediate counter-flux to Ca2+ loss across the tubular system during excitation-contraction coupling.Store-operated calcium (Ca Xaver Koenig et al. demonstrate the physiological activation of store-operated calcium entry in muscle, pointing to a role for this mechanism. The resulting influx of calcium into the cytoplasm is shaped by action potential frequency and calcium pump activity. In contrast, SOCE has evolved to become a fast mechanism in skeletal muscle, probably in parallel with the evolution of the highly organised membrane structures that support the rapid transients of Ca2+ in the fibre evoked by action potentials2. In muscle, the surface plasma membrane of the fibre has regular invaginations, to form the tubular (t-) system. The dominant element of the t-system, the transverse tubules reach each sarcomere to form junctions with the sarcoplasmic reticulum (SR) terminal cisternae3 where the transverse tubular dihydropyridine receptor (DHPR)/L-type Ca2+ channel directly activates the SR ryanodine receptor (RyR) to rapidly activate Ca2+ release4. This process is known as excitation-contraction coupling (EC coupling) and provides the basis for muscle contraction.Store-operated Ca5. The biophysical properties of SOCE in muscle2 suggest its physiological role can be an immediate one, related to compensatory Ca2+ movements during action potential-evoked Ca2+ release. Knowledge of the physiological patterns of store-dependent influx and the conditions associated with its activation are critical in defining physiological role, which will help in our understanding of SOCE-related myopathies, developmental defects and fibre-type shifts in STIM1 and ORAI1 knockout mice10.While the presence of a robust and fast SOCE in muscle is clear, the physiological activation and primary roles of SOCE in this tissue remain elusive2+ within the SR ([Ca2+]SR) is strongly buffered by calsequestrin and the high-capacity SR Ca2+ pumps11. This prevents steady state or tonic activation of SOCE in resting fibres12. Furthermore, the SR strongly buffers Ca2+ during EC coupling, preventing deep depletion of the [Ca2+]SR15, making it difficult to understand how SOCE is activated physiologically in skeletal muscle, if we continue to assume that a bulk depletion of [Ca2+]SR is required for SOCE activation5.Ca2+ release by local depletion of Ca2+ immediately behind the RyRs at the SR terminal cisternae. Local depletions of Ca2+ inside the SR would briefly and uniformly dissociate Ca2+ from the local population of STIM116 to rapidly active SOCE. The rapid transduction of such a signal requires the static junction between the transverse tubules and the SR terminal cisternae membranes, allowing RyR activity to intimately link EC coupling and SOCE2. Consistent with this, it has been demonstrated that only the transverse tubules support SOCE; whereas, the longitudinal elements of the t-system that lack proximity to RyRs have become dynamic Ca2+-buffers, devoid of SOCE17. Here we use a microscopy-based technique with the necessary sensitivity and temporal resolution to measure the occurring Ca2+-fluxes across the t-system membrane19 under physiological stimulation patterns. This approach allowed us to identify that the physiological activation of SOCE is by every action potential-induced Ca2+ release, presenting as a phasic Ca2+ influx. This finding allows the elucidation of the physiological roles of SOCE in muscle and broadens our understanding of the properties of STIM1 and SOCE more generally.We hypothesized that SOCE could be activated during action potential-evoked Ca2+ fluxes across the t-system membrane during action potential-evoked Ca2+ release required imaging Ca2+ in the cytoplasm and t-system with high sensitivity and millisecond temporal resolution. To do this we used mechanically skinned fibres  fibres from the rat, as previously described19. This preparation preserves intact EC coupling20 and respective Ca2+ release and force responses are indistinguishable from those in intact fibres23. The removal of the sarcolemma during the skinning process grants access to the cytosol and allows for the measurement of the intracellular Ca2+ levels with the use of a spectrally separate Ca2+-sensitive dye from rhod-5N, fluo-4.To resolve Ca2+ .Figure 2+]t-sys signals shows a fast decline in t-system Ca2+ associated with each action potential-evoked Ca2+ release  omitted to resolve individual [Ca2+]t-sys depletion events. Still the depletion of [Ca2+]t-sys could be fitted by exponential functions  with respect to [Ca2+]t-sys we obtained a strong linear relationship  suggesting that the observed Ca2+ flux was carried by a Ca2+-selective ion channel24.When we evaluated the t-sys depletion nor its rate of depletion 28. The presence of 10 µM tetracaine allowed action potential-induced Ca2+ release to remain active, though reduced. Furthermore, the use of raised Mg2+ as a RyR antagonist is known not to affect t-system excitability30. Regardless, the presence of the antagonist did not affect the maintenance of excitability as we observed the tonic Ca2+ influx in each case, which likely reflects L-type Ca2+ channel influx31.We are aware that it is possible for ryanodine and tetracaine to affect the DHPR and voltage-gated Na2+ released from the SR into the junctional space per se rather than SR Ca2+ depletion caused activation of phasic t-system Ca2+ flux. To this end we manipulated the [Ca2+] in the junctional space during SR Ca2+ release by bathing the fibre in a solution containing 10 mM BAPTA instead of EGTA. BAPTA provides a much faster rate of Ca2+ chelation. Under these conditions we observed a suppression of the cytosolic Ca2+ transients during EC coupling, as expected, but also observed the persistence of the phasic Ca2+ influx from the t-system . This is consistent with depletion of Ca2+ from within the SR as the trigger for the phasic t-system Ca2+ flux and strongly argues against a mechanism of Ca2+-induced Ca2+ influx, that could have potentially been carried by members of the TRP family of ion channelsAdditionally, we examined the possibility that the Ca2+]t-sys depletion is dependent on the Ca2+ depleted from within the SR.Together, these experiments Figs. -5 clearl2+ flux2. To determine the relationship between the amount of Ca2+ released from the SR and the amount of Ca2+ depleted from the t-system, we determined the total Ca2+ released from the SR under control conditions and under conditions where Ca2+ release was either suppressed or facilitated  there was a constant amount of about 0.14 mM Ca2+ depleted from the t-system (with respect to t-system volume). When SR Ca2+ release surpassed a threshold of 1.2 mM Ca2+ (with respect to SR volume) depletion of the t-system Ca2+ increased with increasing Ca2+ released from the SR. A maximum plateau was reached at a value of about 0.3 mM (right branch of the graph). The amount of extracellular Ca2+ entering the cytoplasm from the t-system was only a small fraction relative to the contribution of the SR during EC coupling. Ca2+ released from the SR contributed about 99% to the Ca2+ entering the cytoplasm  when a certain threshold of SR Ca2+ release was exceeded.To be consistent with the phasic flux being SOCE, the degree of SR depletion should be represented in the magnitude of the t-system Ca2+ pump strongly restricts the activation of SOCE by re-sequestering virtually all of the released Ca2+, eliminating the possibility that SOCE refills the SR following EC coupling5. To test this we directly assessed SOCE during EC coupling in the presence and absence of a functional SR Ca2+ pump. The SR Ca2+ pump was poisoned with cyclopiazonic acid (CPA) added to the cytoplasmic solution to inhibit the uptake of Ca2+. Figure 2+]t-sys to be lowered to a new steady level prior to electrical stimulation. This change in [Ca2+]t-sys occurred in the absence of a change in the t-system electrochemical gradient for Ca2+, indicating that the lowering of [Ca2+]SR to a new steady level upon blocking of the SR Ca2+ pump caused tonic activation of a subpopulation of SOCE channels. Importantly, the commencement of phasic [Ca2+]t-sys depletions with individual electrical stimuli, indicated the additional, rapid recruitment of SOCE channels during the rapid release of Ca2+ from SR. This also indicates that [Ca2+]SR remained high enough in the presence of CPA prior to electrical stimulation to keep a population of SOCE channels inactive. With cessation of electrical stimulation, the lowered [Ca2+]t-sys did not recover in the continued presence of CPA, in contrast to the control . This suggests that high-EGTA suppresses the rise of cytoplasmic [Ca2+]cyto to critically reduce the amount of Ca2+ able to bind to t-system Ca2+-pump binding sites during Ca2+ release to reduce Ca2+ extrusion. In contrast, under weakly buffered conditions, the higher [Ca2+]cyto allows the t-system Ca2+ fluxes to be bidirectional and effectively nullify each other. Taken together, the presence of high EGTA provides Ca2+-buffering in the junctional space to reduce the ability of the t-system to extrude Ca2+, thereby isolating SOCE and the tonic influx of Ca2+ during a train of action potentials that results in the observed [Ca2+]t-sys depletion  a dependence on the amount of Ca2+ depleted from the SR   reion Fig. , 4, (iv)2+ transient of a tetanus15. However, by directly measuring SOCE during EC coupling we show that this is not the case  signal in a resting fibre upon poisoning of the SR Ca2+ pump and thorough depletion of SR Ca2+9. The conclusion was based on the assumption that only the stronger BiFC signal represents prearranged complexes. It is, however, possible that both the full and depleted SR have prearranged Orai1-STIM1 complexes, differing only in absolute number, and therefore BiFC signal.The rapid activation of SOCE in conjunction with action potential-induced Ca1.Our line of argument of pre-coupled Orai1-STIM1 complexes is further supported by the observation that SOCE activated with a time constant of only 10 ms , given the positioning of CSQ at the SR terminal cisternae, immediately behind the RyRs and the unique Ca2+-adsorbing and high Ca2+-buffering capacity of CSQ41. Thus the spatiotemporal separation of Ca2+ sources inside the SR makes the near membrane a privileged signalling environment where Ca2+ can rapidly and locally deplete, with the opening and closing of RyRs. These transient depletions of Ca2+ near the membrane can happen without major depletions of the bulk [Ca2+]SR be it during a spark, twitch or punctuated throughout a tetanus15. This system provides the environment for the rapid dissociation of Ca2+ from STIM1 in the near membrane16 for activation of SOCE following every action potential, as we have demonstrated . While the normal positioning of CSQ and RyRs has been shown46, the exact positioning of STIM1 is unknown because of the lack of a specific STIM1 antibody to use for localization imaging32. Developing a picture of how STIM1 and RyRs position themselves relative to each other at the terminal SR and identifying how this arrangement changes under different conditions will be required to gain a further understanding of signalling at this nano-domain. However, our high-spatiotemporal imaging of Ca2+ transients  muscle was rapidly excised and placed in a Petri dish under paraffin oil above a layer of Sylgard. Small bundles of fibres were exposed to a Ringer solution containing 2.5 mM of the Ca2+-sensitive dye rhod-5N for at least 10 min. Thereafter, segments of individual fibres were isolated and the sarcolemma was mechanically removed with forceps (fibre skinning). The skinned fibres were mounted in a custom built chamber on top of a 1.5 coverslip and bathed in an internal solution (in mM): 90 HEPES, 10 EGTA, 40 HDTA , 8.77 MgO, 1.94 CaCO3, 8 ATP*2Na, 10 creatine phosphate*2Na, with pH adjusted to 7.1 ± 0.1 with KOH and resulting free Ca2+ and Mg2+ concentration of 50 nM and 1 mM, respectively. Fluo-4 was added at a concentration of 10 µM. Electrical field stimulation was delivered by two platinum electrodes running in parallel to the fibre long axis. Individual pulses were applied at various frequencies (0.2–50 Hz) by rectangular voltage steps of up to 100 V in amplitude and 2–4 ms in duration.All experiments on rats were approved by the Animal Ethics Committee at The University of Queensland. Male Wistar and Sprague rats 4–8 months of age  were killed by asphyxiation in CO−1). Pinhole was set to 1.5 AU. Fluorescence intensity across the imaged fibre area was averaged for both colours to derive traces of the mean fluorescence intensity over time that were then converted to [Ca2+]cyto and [Ca2+]t-sys. Calcium concentrations ([Ca2+]), refer to free ion concentrations if not stated otherwise.The experimental chamber was mounted above a ×20 objective of a Leica TCS SP8 laser scanning confocal microscope system equipped with a 12 kHz resonant scanner. Rhod-5N and Fluo-4 were excited by 561 and 488 nm laser lines, respectively, and emitted light was collected with high-sensitivity GaAsP detectors in bands of 40 nm. xyt image series of the two colours were taken in sequential mode with an individual xy image size of 512 × 128 pixels and a time resolution of 18 ms (55 frames sKD) of fluo-4 was determined by measuring the dye fluorescence in the presence of free cytosolic [Ca2+] of 0.05, 0.8, 10 and 2000 µM in 4 fibres. Fluorescence values were normalized to the maximal values and fit with a Hill equation. The so obtained KD-value amounted to 1 µM, which is in good agreement with other reports54. Fluorescence recordings were then transformed to [Ca2+]cyto according to the formulaThe dissociation constant  and neglecting the much smaller endogenous buffering capacity56 the total amount of Ca2+ present can be calculated from the free [Ca2+]cyto according to the following equation:This estimation is based on a method originally described by Melzer et al.And the amount of total calcium released by the SR is obtained by:2+]before and [Ca2+]after are the free calcium concentrations right before and right after each action potential, KD is the dissociation constant of EGTA (200 nM)56, and [EGTA]T is the total EGTA buffer concentration. The so converted values refer to the cytosolic volume of the fibre and were converted to values referring to the SR volume by assuming a cytosol to fibre volume ratio of 0.7 and a SR to fibre volume ratio of 0.09357.where [Ca19. Fluorescence values were converted to [Ca2+]t-sys using equation  was calculated as the difference between the [Ca2+]t-sys right before and immediately after each electrical stimulus. The recovery of [Ca2+]t-sys (∆[Ca2+]t-sys depletion) of the signal between consecutive stimuli was calculated as the difference between the [Ca2+]t-sys right after and immediately before the following electrical stimulus.The change in [Ca2+]t-sys and ∆[Ca2+]SR . All of the so obtained values were then binned into the following intervals of ∆[Ca2+]SR: [0–0.5], [0.5–1], [1–2], [2–3], [3–4], [4–oo] mM. Data were fit with a nonlinear least square routine to a segmented equation consisting of an initial constant baseline followed by an exponential rise according to the formula:To derive the relationship between ∆[Ca]SR Fig. , [Ca2+]tx0 indicating the start of exponential rise.with 2+]t-sys eventually reached a new steady state, a condition in which the single depletion and reuptake steps must exactly balance each other  during repetitive stimulation is the sum of all individual depletion and re-uptake steps, i.e.i and Yri being the ith amplitude of depletion and re-uptake, respectively. The amplitudes of the individual depletions steps followed an exponential, while the amplitudes of the reuptake remained constant 6) yields2+ into the cytosol as determined by the fluo-4 signal the respective depletion of the t-system was only moderately increased reaching a limiting level of about 0.3 mM.On a few occasions electrical stimulation did not result in the generation of only a single action potential but was accompanied by a second action potential or even a small burst like discharge. Also, spontaneous action potentials occasionally seen during experiments happened to sometimes coincide with the electrical stimulation. Although these super events released considerably larger amounts of Cat-test was used. For comparing more than two groups ANOVA with Tukey’s post hoc test was used. Paired testing was applied when the data points to compare were sampled from the same fibres. All data are given as mean ± SEM.For comparing two groups of data a two-tailed Student’s The authors declare that all data supporting the findings of this study are available within the article and its supplementary information files.Supplementary Information(PDF 1903 kb)