KIT, BAALC, ERG, MN1, CDX2, WT1, PRAME, and HOX genes. High expression of these genes constitutes usually an unfavorable prognostic factor. Upregulation of FLT3 and NPM1 genes, independent on their mutation status, was also reported in AML and correlated with poor outcome. However, transcriptome is not limited to the protein-coding genes; other types of RNA molecules exist in a cell and regulate genome function. It was shown that microRNA (miRNA) profiles differentiated AML groups and predicted outcome not worse than protein-coding gene profiles. For example, upregulation of miR-10a, miR-10b, and miR-196b and downregulation of miR-192 were found as typical of AML with NPM1 mutation whereas overexpression of miR-155 was associated with FLT3-internal tandem duplication (FLT3-ITD). Development of high-throughput technologies and microarray replacement by next generation sequencing (RNA-seq) enabled uncovering a real variety of leukemic cell transcriptomes, reflected by gene fusions, chimeric RNAs, alternatively spliced transcripts, miRNAs, piRNAs, long noncoding RNAs (lncRNAs), and their special type, circular RNAs. Many of them can be considered as AML biomarkers and potential therapeutic targets. The relations between particular RNA puzzles and other components of leukemic cells and their microenvironment, such as exosomes, are now under investigation. Hopefully, the results of this research will shed the light on these aspects of AML pathogenesis which are still not completely understood.The last two decades of genome-scale research revealed a complex molecular picture of acute myeloid leukemia (AML). On the one hand, a number of mutations were discovered and associated with AML diagnosis and prognosis; some of them were introduced into diagnostic tests. On the other hand, transcriptome studies, which preceded AML exome and genome sequencing, remained poorly translated into clinics. Nevertheless, gene expression studies significantly contributed to the elucidation of AML pathogenesis and indicated potential therapeutic directions. The power of transcriptomic approach lies in its comprehensiveness; we can observe how genome manifests its function in a particular type of cells and follow many genes in one test. Moreover, gene expression measurement can be combined with mutation detection, as high-impact mutations are often present in transcripts. This review sums up 20 years of transcriptome research devoted to AML. Gene expression profiling (GEP) revealed signatures distinctive for selected AML subtypes and uncovered the additional within-subtype heterogeneity. The results were particularly valuable in the case of AML with normal karyotype which concerns up to 50% of AML cases. With the use of GEP, new classes of the disease were identified and prognostic predictors were proposed. A plenty of genes were detected as overexpressed in AML when compared to healthy control, including Acute myeloid leukemia (AML), the most frequent leukemia in adults, is a severe myeloproliferative disorder with the high risk of relapse and high mortality rate , 2. Rand ex vivo drug sensitivity in a cohort of over 500 AML patients , a rare aberration occurring in about 2% of all AML cases, as a sole abnormality or accompanied by t, t, or other cytogenetic aberration . TranscrMixed-phenotype acute leukemia (MPAL), a rare type of progenitor leukemia with ambiguous expression of myeloid and lymphoid lineage markers, was studied by Pallavajjala et al. (2018) . RNA-seq RUNX1–RUNX1T1 fusion, typical of t AML, the patient harbored two additional translocations with the contribution of VPS13B gene, a causative gene of Cohen syndrome, encoding vacuolar protein sorting 13, forming TM7SF3–VPS13B and VPS13B–RUNX1 fusion genes.Being a useful tool for fusion transcript detection, RNA-seq was applied to identify a complex 3-way translocation t in an individual AML M2 patient . In addi CIITA-DEXI fusion transcript, occurring in three isoforms, was found in 48% of NK-AML cases. Of note, the maximal number of fusion transcripts found in one NK-AML case was 57. Although some fusions were generated posttranscriptionally, these results suggest that genome-level changes are not so rare in AMLs with normal karyotypes. The significance of particular fusions remained to be elucidated.With the use of a whole transcriptome sequencing, 88 new fusion transcripts were discovered in AML by Wen et al. . In tota NUP98-PHF23 [ CBFA2T3-GLIS2 [ CBFA2T3-GLIS2 fusion resulted from a cryptic inversion of chromosome 16 and was correlated with high risk of relapse and poor outcome [ CBFA2T3-GLIS2-positive patients [DHH), and Ras Homologue Enrich in Brain Like 1 (RHEBL1) gene, coding for a small GTPase of the Ras family. DHH-RHEBL1–positive patients exhibited a specific gene expression pattern, with upregulation of FLT3, BEX1, MUC4 and AFAP1L2 genes. The outcome of these patients was even worse than that of the patients with exclusive CBFA2T3-GLIS2-rearrangement. Notably, targeted treatments against AML with CBFA2T3-GLIS2 are under evaluation. GANT61, the most potent inhibitor of GLI family proteins, which are the final effectors of Hedgehog pathway, seems to be efficient also against GLIS2 chimeric proteins [Also in pediatric CN-AML, novel fusion transcripts were identified with RNA-seq, e.g.,98-PHF23  or CBFA2T3-GLIS2 , 168, oc outcome , 168. Sc outcome  demonstrpatients . The novproteins . AML1 gene, at present termed RUNX1. Both transcripts, AML1a and AML1b, shared a runt homology domain, responsible for DNA binding, whereas transcriptional activation domain was present only in AML1b. The authors found that the two AS products regulated hematopoietic myeloid cell differentiation in an antagonistic way, presumably via competing for the binding to CBF2B gene encoding transcriptional activator. While AML1a inhibited granulocytic differentiation and induced cell proliferation upon granulocyte colony-stimulating factor (G-CSF) treatment, concomitant AML1b overexpression recovered the granulocytic differentiation.The role of alternative splicing (AS) in AML pathogenesis was first highlighted by Tanaka et al. (1995)  who anal SRSF1 was associated with solid tumor promotion, mutations in genes encoding splice factors, i.e., SF3B1, SRSF2, U2AF1, are considered as important drivers of hematological disorders such as MDS and AML [ FLT3, aberrant splicing of BCL2 gene, linked with drug resistance, and overexpression of WT1 and E2F1 genes, which encode transcription factors taking part in AS regulation [ NPM1 gene are upregulated in AML and ALL at diagnosis, decrease in CR and increase again at relapse [ NPM1 gene isoforms was significantly associated with shorter overall and disease-free survival. This suggested that not only mutation but also expression level of NPM1 gene affects patient outcome. Aberrant proportions of particular NPM1 splice variants could be linked to abnormal expression of genes encoding alternative splicing factors.Development of NGS contributed to the progress in AS research. Now, the role of splicing abnormalities in AML progression and drug resistance is incontestable . While o and AML . Contribgulation . We rece relapse . High exRNA-seq of two samples collected from an individual AML M2 patient at diagnosis and remission were explored in the context of AS by Li et al. (2014)  and Gao  U2AF1 mutation in a transgenic mouse model [ U2af1-mutated mice revealed altered hematopoiesis and changes in premRNA splicing. Comparing the results of the analysis with human RNA-seq data from TCGA AML cohort displayed enrichment of U2AF1-induced splicing alterations in processing genes, ribosomal genes, and recurrently mutated MDS and AML-associated genes . The authors concluded sequence-specific AS pattern triggered by mutant U2AF1 was similar in mouse and human cells.Shirai et al. (2015) investigated the consequences of the most commonse model . Whole t KMT2A rearrangements, were found in one cluster, together with one CML cell line. Deregulated genes associated with this particular cluster were involved in mRNA splicing via spliceosome. The obtained results corroborated contribution of epigenetic-mediated splicing events to progression of KMT2A-AML and associated alternative splicing mediated by K79me methyltransferase, encoded by DOT1L gene, with leukemogenesis.Li et al. (2018) integrated AS events derived from RNA-seq with H3K79me2 ChIP-seq data across 34 human normal and cancer cell types . Cluster PML-RARA transcripts with exon junctions not described earlier in t, and expression changes with time and treatment. Other chRNAs originated from two adjacent genes , nonadjacent exons separated by thousands of base pairs , two distant genes , or gene fragments corresponding to opposite chromosome strands. Extending their research to a larger AML patient cohort and normal CD34+ transcriptome, the authors identified four new types of tumor-specific chRNAs recurrently expressed in AML samples . On the basis of PML-RAR fusions, it was shown in one patient several isoforms of the same chimeric transcript can coexist and present various sensitivity to therapeutic agents. The resistance to treatment can be indicated by the appearance or increase of fusion transcripts. However, the oncogenic potential of chRNAs needs to be verified by further research.Apart from well-established fusions and alternative transcripts, another class of transcripts has been recently discovered in tumor as well as in normal cells by RNA-seq , 176. Th miR-233. In AML, it represented almost 50% of all miRNA reads. miRNAs, which displayed differential expression between AML and control CD34+ pool, included miR-362-3p and miR-25, overexpressed and underexpressed in AML, respectively. Comparison of NGS-based miRNA profiling with the array- and RT-PCR-based approaches, showed microarray and real-time analyses underestimated the expression of some miRNA, although the general correlation between platforms was significant. The authors did not find acquired mutations in miRNA genes but revealed several novel germline polymorphisms. Comparing the results of miRNA expression with the sequence of this particular AML patient genome, which was known earlier [ TNFAIP2 and proved this mutation generated a new miRNA binding site. As this mutation resulted in a Dicer-dependent translational repression of a reporter gene, the consequence could be a translational repression of TNFAIP2, previously described as a target of PML-RARA or PZLF-RARA fusion genes and highly expressed in hematopoietic cells. TNFAIP2 mutation was predicted to generate imperfect binding sites for miR-223 and miR-181b, but the experiments conducted on AML cell lines did not confirm contribution of miR-223 and miR-181b and any other known miRNA to the translational repression of mutant TNFAIP2 3′-UTR. The authors did not exclude possibility of regulation by a new, unknown yet, miRNA. Nevertheless, the TNFAIP2 3′-UTR mutation must be rare as it was not found in any other AML samples from 187 patients screened.Compared to microarrays, NGS enabled more comprehensive and quantitative analysis of miRNome. Ramsingh et al.  analyzed earlier , they id miR-370, often upregulated in AML, was proven to target tumor suppressor NF1, downregulated in more than 30% AMLs. Because NF1 suppression activates RAS similarly as RAS-activating mutation, AML patients with miR-370 overexpression may potentially benefit from additional treatment with either RAS or mTOR inhibitors.Integrating SNP and mRNA arrays with microRNA profiling of 16 myeloid cell lines, García-Ortí et al.  associat miR-145 and miR-146a, localized in a commonly deleted in AML region 5q, initiated leukemia in mice. Using small RNA sequencing they identified 28 novel miRNAs, 18 of which mapped to leukemia-associated copy-number alterations, and may play a role in leukemogenesis.Starczynowski et al.  who glob RUNX1T1 in miRNA binding. The so called competing endogenous RNAs (ceRNAs) cross-regulate each other by competing for binding to shared miRNAs. The predicted set of ceRNAs contained multiple oncogenes and members of the integrin, cadherin, and Wnt signaling pathways. One-third of those genes were differentially expressed between t AML and normal granulocyte-macrophage progenitor cells. Taking into account experimentally validated miRNA binding sites, the authors selected 21 top RUNX1T1 ceRNAs, including 13 which shared miRNA binding sites with RUNX1T1, e.g., PLAG1, TCF4, NFIB, and YWHAZ. Therefore, the authors supported the hypothetical miRNA sponge function of RUNX1T1 gene, particularly its 3'UTR, present in a leukemic RUNX1-RUNX1T1 fusion transcript and overexpressed up to 1000 times in t versus other and control samples.Using t AML mRNA- and miRNA-sequencing data from TCGA project, Junge et al.  construc PIWIL4 gene, encoding one of PIWI proteins, in AML samples, the highest in AML with KMT2A-AF9 translocation conferring poor prognosis. Of note, prognostically favorable AML with PML-RARA and inv16 showed the lowest levels of PIWIL4. Overexpression of PIWIL4 was correlated with high expression of genes involved in cell proliferation, such as FLT3, CBL and NRAS. Contrary, depletion of PIWIL4 in AML with KMT2A rearrangements drastically reduced leukemic cell growth in vitro and in vivo, but did not affect normal cord blood CD34+ cell growth. Out of 10 thousand unique piRNAs detected in wt THP-1 cells, over 1000 revealed changed expression following PIWIL4 knockdown, including 80 mapped to genes indicated by RNA-seq as deregulated by PIWIL4 depletion. Interestingly, most of these genes belonged to the actin cytoskeleton regulation pathway.From small RNAs, miRNAs are definitely best characterized and their association with normal and malignant development is well established. Recently, a little longer (25-33 nt) P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), responsible for epigenetic silencing of transposable elements (TEs) in germline tissue, have been correlated with brain functioning and tumor transformation , 182. AbThe role of TEs in AML and MDS was highlighted by Colombo et al. . While h DLK-DIO3 and SNURF/SNRPN loci, are expressed in a lineage- and developmentally restricted manner in human hematopoiesis. Moreover, 120 snoRNAs, including SNORA21, SNORA36C and SCARNA15, displayed consistent differential expression in AML, and, what is even more interesting, all of them were decreased in AML samples compared to normal CD34+ cells. Of note, although the majority of snoRNAs were embedded in the introns of host genes, expression levels of snoRNAs did not correlated with expression or alternative splicing of host genes, which suggested cellular levels of mature snoRNAs were determined by other factors. No somatic mutations were detected in the snoRNA genes, either. By the way, the authors found a few novel snoRNAs and proved standard transcriptome sequencing cannot reliably distinguish unspliced primary host gene RNA from correctly processed snoRNA [Small nucleolar RNAs (snoRNAs) are basically involved in the posttranscriptional modification of ribosomal RNAs, in cooperation with protein partners , 187. Ind snoRNA . FLT3-ITD and mutations in such genes as NPM1, CEBPA, IDH1, IDH2, ASXL1, and RUNX1. For example, patients with mutated NPM1 revealed upregulation of several antisense transcripts of HOX genes , plasmacytoma variant translocation 1 (PVT1), and the coiled-coil domain containing 26 (CCD26) lncRNAs. Wilms tumor 1 antisense RNA (WT1-AS) lncRNA was found as typical of FLT3-ITD signature whereas downregulation of HOXB-AS3 lncRNA was noted in CEBPA-mutated AML. RUNX1 mutation was associated with the increase of lncRNAs located in the proximity of lymphoid marker genes , the immunoglobulin heavy locus (IGH) complex, and vault RNA 1-1 (VTRNA1-1), which was linked with multidrug resistance. Of note, no specific lncRNA profiles were found for DNMT3A and TET2 mutations which frequently occur in older CN-AML patients. Instead, prognostic signature composed of 48 lncRNAs was identified, indicating correlation of lncRNA expression with AML treatment response and survival.A novel class of functional RNAs, which do not encode proteins and are longer than 200 nt, are referred to as long noncoding RNAs (lncRNAs) . Their r HOTAIRM1, granulocyte-specific lncRNA, was associated with inflammatory and innate immune response pathways, and was strongly correlated with genes upregulated in AML with NPM1 mutation. In addition, novel ncRNA regulators of granulopoiesis were predicted, e.g., LINC00173 expressed specifically in mature granulocytes and negatively associated with the expression of genes related to stemness, cell cycle progression and cancer. The role of LINC00173 in granulocyte proliferation and differentiation was then confirmed by its transcriptional repression with CRISPR-interference (CRISPRi) in NB4 leukemia cell line. Similarly, gain- and loss-of-function experiments validated the function of miRNAs and lncRNAs of the human DLK1-DIO3 locus in the differentiation and maintenance of megakaryocytes. Transcriptome analysis of 46 pediatric AML samples allowed identification of prognostically relevant ncRNA signatures shared by normal HSCs and AML blasts of distinct cytogenetic and morphologic subgroups.In 2017, Schwarzer et al.  presente in vivo. Some of them seemed to promote leukemia stem-cell signatures, e.g., Pvt1 and Lilam, whose functions were correlated with the function of MYC oncogene. Another lncRNA, termed Pilna, was required for the myeloid lineage during bone marrow reconstitution.In the same year, a systematic analysis of lncRNAs in hematopoiesis and hematological malignancies was also conducted by Delás et al.  with a m MEG3) and other FAB subtypes comparing to normal CD34+ cells. Reanalysis of 200 transcriptomes from TCGA AML dataset was also used to construct prognosis-related lncRNA module pathway network [Transcriptome analysis of an individual primary AML sample from TCGA dataset enabled discovering 194 unannotated small RNAs in the 17-35 nt size range, 258 unannotated small RNAs in the range of 36-100 nt, and 977 previously unannotated multiexon lncRNA transcripts [ network . First,  CEBPA, NPM1, FLT3-ITD and TP53 mutation was noted for particular subtypes. Despite some similarities between lncRNA and mRNA expression, both types of transcripts stratified AML patients in different ways. This suggested a limited overlap in information retrieved from various levels of transcriptome analysis and underpinned the rationale for further lncRNA studies.Relation between lncRNA expression and AML prognosis was also studied by Mer et al.  who sequ DANCR (Differentiation Antagonizing Nonprotein Coding RNA), lincRNA highly conserved between mice and humans, overexpressed in hepatocellular carcinoma. DANCR, described as a regulator of the Wnt pathway, crucial for the biology of LSCs, was proved to play a role in LSC self-renewal and quiescence. Decreased expression of MYC and other genes from Wnt pathway in AML cell line after DANCR knockdown confirmed association of DANCR with Wnt pathway.In 2019, Bill et al. , while a KMT2A transcripts, including circular isoforms, resulted from exon scrambling was shown by Caldas et al. in 1998 [Though most human and mammalian premRNAs are spliced into linear molecules, the existence of circular RNAs (circRNAs), generated by noncanonical splicing , was sporadically reported within the last 30 years . In leuk in 1998 . The abu circ_EMB, generated from EMB gene encoding embigin, transmembrane protein from the immunoglobulin superfamily considered as a cancer biomarker, was highly abundant in APL. Upregulation in CN-AML was noted for circ_SMARCA5, originated from SMARCA5 gene encoding a core component of chromatin remodeling and spacing factor RSF, which promotes cell proliferation. In compliance with the theory of circRNA functioning as miRNA sponges, the authors found miR-10b (a member of the miR-99 family) binding site on circ_SMARCA5. As SMARCA5 expression was shown to be affected by miR-99, binding of miR-10b by circ_SMARCA5 could relieve linear SMARCA5 transcripts from repression and contribute to the accumulation of undifferentiated myeloid cells. Analysis of data from a larger AML cohort revealed that the gene loci frequently mutated in AML produced significantly more circRNAs.You and Conrad who elaborated the acfs algorithm for identification and quantification of circRNAs from single- and paired-ended RNA-seq data , demonst NPM1 gene have been extensively studied by Hirsch et al. [ NPM1 mutation), one CML cell line, and 3 samples derived from the PBMC fraction of healthy volunteers, 15 circular NPM1 transcripts were identified, including those previously deposited in circBase and novel ones. Oxford Nanopore technology of long read sequencing was harnessed to verify the internal structure of identified transcripts. One of them (hsa_circ_0075001), which exhibited highly differential expression in the AML cell lines, was quantified in a cohort of 46 AML patients. Based on hsa_circ_0075001 expression, patients were divided into two groups that differed in the expression of more than 2000 other genes. AML patients with high hsa_circ_0075001 expression, presented upregulation of ribosomal protein genes, increase of total NPM1 expression, and downregulation of genes involved in the Toll-like receptor (TLR) signaling pathway and genes targeted by miR-181 , which is deregulated in CN-AML. The expression of hsa_circ_0075001 correlated with total NPM1 expression, but was not affected by the NPM1 mutational status. Global analysis of circRNA expression in 10 AML samples and 10 sorted cell fractions form healthy hematopoietic controls evidenced the existence of circRNA transcripts for almost half of all highly expressed genes [ circFLT3 expression was not correlated with FLT3 gene expression in AML samples . AML patients and healthy controls differed in the expression levels of circRNAs arisen from 27 genes, including ANGPT1, UGCG and FLT3. In addition, AML subgroup-specific circRNA signatures were identified, e.g., NPM1-mutated patients could be distinguished from NPM1-wt patients based on their global circRNA expression (but not based on circNPM1 expression).In 2017, circRNAs generated from circPVT1, a circular transcript of PVT1 gene in the studied AML cases compared to NK-AML. The expression of circPVT1 was correlated with PVT1 gene copy-number increase and high PVT1 expression in AML patients with MYC amplification.L'Abbate et al.  who anal circPAN3, was overexpressed not only in THP-1/ADM cells but also in refractory and recurrent AML samples. Silencing of this circRNA restored sensitivity to doxorubicin in THP-1/ADM cells, which indicated its significant role in chemoresistance mediation.Contribution of circRNA to drug resistance was recently shown by Shang et al. with the use of doxorubicin-resistant THP-1 AML cell line (THP-1/ADM) . The autBIK, CCNA1, FUT4, IL3RA, HOMER3, JAG1, WT1) and 6 downregulated . Moreover, the expression levels of WT1, FUT4, CCNA1, HOMER3, JAG1, TRIB2, and SERPINB9 were strongly associated with FAB classification whereas WT1, JAG1, ALDH1A1, TRIB2, and PLXNC1 with cytogenetics. For example, WT1 was the most overexpressed in AML with inv(16) or t, CCNA1 in t, while BIK expression was absent or extremely low in t. 7 upregulated genes were also measured in pediatric AML, where BIK, FUT4, and WT1 showed the most significant increase in expression.Leukemic blasts which accumulate in BM and PB of AML patients represent cells blocked at a particular stage of differentiation. However, in the majority of GEP studies performed within the first years of microarray technology development, bulk AML cells were compared to the whole pool of mononuclear cells from healthy control samples. Then, the cell sorting techniques were introduced to select particular cell fractions. The example of a study where AML cells were compared not only to unselected healthy BM and PB samples, but also to normal hematopoietic CD34+ cells extracted from BM and PB, was that of Stirewalt et al. . They foAfter the discovery of the self-renewing leukemic stem cells (LSCs), the main focus was shifted to these early progenitor cells capable of initiating leukemogenesis. How they are different from normal human hematopoietic stem cells (HSCs) was first shown in 2009 by Majeti et al.  who iden VNN1, RBPMS, SETBP1, GUCY1A3, MEF2C, and HOPX. Genes associated with proliferation, cell cycle, and differentiation were systematically repressed in the LSCs. The identified LSC gene expression signature (LSC score), reflecting self-renewal ability, was validated on four independent datasets of 1047 patients in total, leading to similar conclusions. OS, EFS, and RFS were worse for patients with high LSC score [Gentles et al. showed the correlation of high expression of leukemic stem-cell genes with adverse outcomes in AML . CompariSC score . FLT3-ITD and adverse cytogenetics. The LSC score was validated by xenotransplantation assays and by reanalysis of microarray and RNA-seq data from five independent cohorts of more than 900 AML patients with different subtypes. Comparing with other prognostic determinants such as age, WBC count, cytogenetic risk group, and mutational status, LSC17 score was the strongest and independent prognostic factor. In the end, the authors designed a custom NanoString assay to easily analyze expression of 17 genes from the LSC signature. The assay should allow for rapid risk assessment at diagnosis, application of more intensified investigational therapies in the case of high-score patients, and protection of low-score patients against unnecessary toxicity. To test its efficiency in childhood leukemia, Duployez et al. applied the LSC17 score to 228 de novo pediatric AML patients [ RUNX1 mutations, high LSC17 score gained the best statistics.Ng et al.  comparedpatients . Indeed,patients . Among a S100A8, SOD2, and IGFBP7 [ IGFBP7, encoding insulin-like growth factor-binding protein 7, in LSCs was correlated with reduced sensitivity to chemotherapy. Restoration of IGFBP7 expression by a recombinant human gene resulted in differentiation, inhibition of LSC survival, and improved response to therapy, without affecting normal hematopoiesis and HSC survival. Therefore, IGFBP7 gene was postulated to be a factor responsible for the persistence of LSCs.Recently, GEP of LSCs, HSCs, and leukemic progenitors from the same AML bone marrow enabled identifying three genes overlapping in the results of two pairwise comparisons (LSCs vs. HSCs and LSCs vs. leukemic progenitors):d IGFBP7 . Signifi In vitro experiments with cell line models are convenient, but have also some limitations. In fact, animal models are the best option to study particular gene function, consequences of early pathogenic events or treatment with potential therapeutics. Using retroviral insertion mutagenesis in mice, Erkeland et al. (2004) [ STAT genes, were implicated in cytokine signaling. Another network, consisted of gene expression regulators, was able to discriminate between AML patients with favorable and unfavorable prognosis. In the unfavorable group, HOXA9, MEIS1, and CCND3 genes were increased whereas BCOR and GFI1 genes were decreased.Deep insight into the biology of the disease is not possible by the analysis of cells extracted from patient BM or PB.. (2004)  identifi. (2004)  and 130 . (2004) . First,  MECOM (previously EVI1) target genes by comparison in MECOM-overexpressing murine myeloid leukemia cell lines  before and after shRNA-mediated MECOM knockdown. MECOM, oncogenic transcription factor associated with human myeloid malignancies of poor prognosis, is overexpressed in 8–10% of adult AML and up to 27% of pediatric leukemias with KMT2A rearrangements. MECOM-induced leukemic cells present impaired myeloid differentiation, resistance to apoptosis, and aberrant cell cycle regulation, which results in excessive proliferation. Combining RNA-seq expression profiling with ChIP-seq, the authors found MECOM directly bound to and downregulated Cebpe gene, encoding a master myeloid differentiation regulator, Serpinb2 gene, encoding serine protease inhibitor involved in cell cycle regulation, and numerous genes from the Jak-Stat signaling pathway that drive cellular differentiation. In addition, several P2X purinoceptors, responsible for ATP mediated apoptosis in neutrophils and macrophages, appeared to be significantly downregulated in MECOM leukemic cells.Glass et al.  used NGS Meis1 and Hoxa9 genes in the purified fetal liver (FL) cells. Both clones differed in the hematopoietic stem-cell frequency and gene expression profile. Considering microRNAs, only a few miRNAs were really highly expressed; more than 95% of all miRNA transcripts came from the top 15 miRNAs. Functional annotation analysis showed the most differentiating genes between Meis1- and Hoxa9-overexpressing clones were related to immune system development, hemopoietic or lymphoid organ development, hemopoiesis, and myeloid cell differentiation. Both, differential expression and/or differential splicing, were observed in the studied transcriptomes. Moreover, hundreds of single nucleotide variants (SNVs), shared by both cell clones or unique for one of them, were identified with respect to the public reference sequence. The study revealed also a number of unannotated transcribed elements.An interesting approach was applied by Wilhelm et al. (2011)  who geneHOX) genes, PBX3, and MEIS1. Based on overall SE distribution, patients were classified to 6 subgroups, including 4 enriched in NPM1 and FLT3 mutation, and one containing all AML samples with KMT2A translocation. The authors concluded KMT2A translocations might induce a unique epigenetic state affecting overall enhancer landscape, which is consistent with the association of KMT2A fusion proteins with DOT1L deregulation and, consequently, aberrant histone H3K79 methylation. Moreover, patients classified to different SE-clusters showed differences in OS. Discovery of RARA-associated SE which differentiated patient samples and correlated with significantly higher RARA expression in 25% patients, prompted the authors to test the sensitivity of AML cells presenting a strong SE at the RARA locus to RARα targeted compounds. The results of tests conducted on AML cell lines, AML patient derived mouse xenograft models and AML cells ex vivo, demonstrated that RARα agonist SY-1425 (tamibarotene) selectively inhibited the proliferation of AML cells with high (but not low) RARA expression, stimulating the expression of genes linked to granulocytic differentiation  and retinoic acid response . In the context of efficiency, comparison of SY-1425 to ATRA, used clinically for APL therapy, showed the prevalence of the tested compound. Transcriptional response of APL to retinoids was similar to the response of AML cell lines with high RARA expression to SY-1425. The authors presented the following interpretation of the observed results: while APL-specific PML-RARA fusion protein represses transcription of differentiation-related genes, in non-APL AML, RARA-associated SE induces overexpression of unliganded RARα which acts as a transcriptional repressor of genes regulating by RARα. The practical consequence of clinical significance is that some non-APL AML patient may benefit from the ATRA-like therapy, and increased levels of RARA mRNA could be used as a prerequisite for the treatment by SY-1425, which has a power to reset RARα transcriptional activity.McKeown et al. (2017)  matched  sPRDM16 in AML progression was demonstrated by Dong & Chen on leukemic cell lines [ PRDM16, previously termed MEL1, encodes transcription factor acting as a H3K9me1 methyltransferase responsible for maintenance of heterochromatin integrity. However, only protein isoform deprived of the histone methyltransferase domain, encoded by a short transcript variant, sPRDM16, was associated with AML pathogenesis. The authors showed that SUMOylation of sPRDM16 changed the expression of genes implicated in wound response, cell proliferation, chemotaxis, differentiation, and cell cycle progression, including 13 genes  involved in proliferation and differentiation of hematopoietic and leukemic cells.Not only genetic and epigenetic factors contribute to AML pathogenesis; initiation, progression and maturation of AML are also affected by posttranslational modification of proteins. The important role of SUMOylation ofll lines . PRDM16, CD34-positive, or HLA-DRA- and CD117-positive. Moreover, two of them were outliers in PCA. Complementary targeted DNA sequencing revealed the presence of mutations in DNMT3A and NPM1, and FLT3-ITD in the analyzed AML sample. However, RNA-seq identified only DNMT3A mutation in only one single cell. Possible explanations are not sufficient coverage , imperfect algorithms for mutation detection in RNA-seq data, and mutational heterogeneity of blast cells. An argument for the last explanation can be found in the report of Shlush et al. [ DNMT3A mutation at high allele frequency, but did not detected concomitant NPM1 mutation. Therefore, the authors concluded DNMT3A arose early in AML evolution. The preliminary experiences with single-cell sequencing show this technology demands optimization, but seems to be a strategy of future.Considering AML as a multiclonal cancer, we should be aware of the fact that bulk RNA sequencing reflects what is going on in a dominant clone that is not necessarily a clone of origin. Even application of cell sorting technique may not be sufficient to catch the whole AML heterogeneity as far as we sequence a pool of cells. A promising approach, which can overcome this problem is single-cell gene expression profiling. A pilot single-cell AML study was performed on twenty CD45-positive cells collected from an individual AML patient . GEP shoh et al.  who iden in vitro diagnostic and prognostic microarray supporting the choice of optimal therapy strategy [As a result of years of transcriptome-scale studies, a few commercial tools dedicated to AML and other hematologic malignancies were developed. Affymetrix technology, which conquered the microarray market, was used by SkylineDx (https://www.skylinedx.com), high-tech commercial-stage biotech company headquartered in Rotterdam, the Netherlands, to design AMLprofiler, a qualitativestrategy . AMLprof in vitro diagnostic testing. Although AML-dedicated kits do not exist for MiSeqDx, the system is customizable and a number of partners collaborates with the company to develop new clinical assays. At present, kits for target sequencing are available for other Illumina systems, e.g., TruSight Myeloid Sequencing Panel, targeting 54 genes, including CEBPA, NPM1 and FLT3-ITD, or AmpliSeq for Illumina Myeloid Panel, targeting 40 DNA genes, 29 RNA fusion driver genes, and 5 gene expression levels associated with myeloid cancers, including AML.Illumina (https://www.illumina.com), the company headquartered in San Diego, California, USA, who practically monopolized the NGS market, released the MiSeqDx system, the first FDA-regulated, CE-IVD-marked, NGS platform forOther integrated diagnostic platforms are being developed and validated, e.g., rapid and sensitive NGS-based assay combining karyotyping and mutational screening of AML . Here, tWithin the last two decades, an explosion of AML studies, driven by technological progress, could be observed. AML picture emerging from transcriptome research is very complex and dynamic. AML transcriptome, affected by cytogenetic and genetic variability, resembles a mosaic, composed of many elements interacting with each other. AML subtypes present unique patterns of protein-coding gene and microRNA expression. Moreover, a spectrum of fusion genes, alternative transcripts and newly discovered chimeric RNAs, as well as a lot of noncoding RNAs, including long linear and circular forms, contribute to the complexity of leukemic transcriptome.Transcriptome data cannot be interpreted separately from genetic and genomic data. Even a sole point mutation can affect expression of numerous genes, e.g., when it occurs in a transcription factor, epigenetic regulator, or a crucial member of a signaling pathway. Therefore, genome, exome, methylome sequencing, or targeted resequencing is often combined with RNA-seq. In some cases transcriptome analyses helped to define which mutation was the driver one. It must be remembered that a disease is not limited to leukemic blasts. Recent studies on BM microenvironment underlined its role in AML initiation, progression, and relapse. Recognition of molecular interplay between LSCs and BM niche not only is necessary to understand the AML biology but also opens novel AML treatment directions.Many reports proved the power of global transcriptome profiling and proposed application of GEP for AML diagnosis and prognosis. A single microarray-based test usually classified AML subgroups properly without any a priori knowledge. The following studies utilized more advanced generations of microarrays, composed of an increasing number of probes, and included more numerous cohorts of patients. Of note, the results obtained by different groups were largely overlapping. As gene expression-based prediction of the major cytogenetic subgroups was efficient in both, pediatric and adult, AMLs, GEP appears as an attractive alternative to classical cytogenetics which is laborious and time-consuming. On the other hand, there is no need to harness high-throughput technology when simple, e.g., PCR-based, diagnostic tests can easily confirm the presence of the fusion genes.Because some genes are significantly overexpressed in AML, not only in leukemic blasts but also in BM microenvironment, and their expression affects treatment response, transcript measurement at the time of diagnosis should be obligatory in AML diagnostics. In my opinion, reliable quantitative PCR techniques, e.g., ddPCR, have currently the highest potential to be routinely applied in clinical practice to analyze selected transcript levels. To analyze higher number of transcripts, gene expression arrays or RNA-seq may be applied instead. Although both high-throughput GEP approaches demand specialized, expensive equipment, and well-trained staff, RNA-seq seems to be a superior technology, offering more information at a comparable cost. Within the last few years, small personal, relatively cheap, and portable sequencers started to appear which makes NGS-based tests more available. Moreover, alternative, third-generation sequencing technologies are becoming more and more popular. I believe that future clinical diagnostic laboratories will offer NGS services, not limited to mutation detection, as a standard. Even if accepting GEP as the only diagnostic test would be difficult, it could at least serve as a first screening or a complementary tool.Summarizing, although gene expression studies were not directly translated into clinical practice up to date, they helped us to understand the biology of tumors and undoubtedly contributed to the improvement of classification of hematological malignancies and risk estimation, which is crucial for optimal treatment decision and directs us towards personalized medicine.


The number of circulating platelets as well as the correct aggregability is essential for an appropriate hemostasis. In addition to their primary function, platelets also play a relevant role in a diversity of events, such as chronic and allergic inflammations through the exocytosis of platelet granules. Platelet function, either adhesion, aggregation or secretion, is tightly regulated by a number of cellular mechanisms, among which the mobilization of intracellular free Ca2+ concentration by the release of Ca2+ from intracellular Ca2+ stores, mostly the dense tubular system and acidic lysosomal-like organelles, and the influx of extracellular Ca2+ through plasma membrane channels. A major mechanism for agonist-induced Ca2+ influx in platelets is store-operated or capacitative Ca2+ entry [2+ entry (SOCE) is a mechanism for Ca2+ influx whereby the discharge of intracellular Ca2+ stores secondarily activates Ca2+ influx across Ca2+-permeable channels. A key molecular element of SOCE is the endoplasmic reticulum Ca2+ sensor, STIM1 [2+ store depletion results in the activation of two types of STIM1-regulated channels, named the Ca2+ release-activated Ca2+ (CRAC) channels and the store-operated Ca2+ (SOC) channels. CRAC channels are highly specific for Ca2+ and are composed of Orai1 subunits, while SOC channels are cation permeable channels that consists of still uncharacterized heteromeric complexes of Orai1 and TRPC1 subunits [ICRAC currents in platelets is uncertain but we have reported a functional role for the STIM1-Orai1-TRPC1 ternary complex in intracellular Ca2+ homeostasis in these cells [Platelet agonists increase cytosolic free-Ca2+ entry . Store-osubunits . The exise cells .2+ entry. According to the role of the membrane cytoskeleton as a clamp for constitutive SOCE, we have previously reported that cofilin, an actin-binding protein, is involved in rapid actin filament depolymerization induced by physiological agonists that occurs in a subsecond time scale and, therefore, prior to the detection of SOCE [2152 by protein kinase A upon Ca2+ store depletion and in a Ca2+-dependent manner, a post-translational modification that is essential for the interaction of filamin A with STIM1 [2152 phosphorylation or attenuation of filamin A expression using RNAi impairs its interaction with STIM1, which results in a significant increase in thapsigargin-induced Ca2+ entry, thus indicating that filamin A is a negative modulator of SOCE. In an attempt to ascertain the mechanism underlying the regulatory role of filamin A we found that filamin A knockdown results in an enhancement of the STIM1 interaction with its partner, Orai1, which strongly suggests that filamin A participates in fine-tuning the STIM1-Orai1 interaction in order to generate physiologically adequate Ca2+ signals. In the absence of filamin A, an exacerbated STIM1-Orai1 interaction occurs in response to receptor stimulation, leading to aberrant Ca2+ influx that might result in abnormally enhanced platelet responses. In fact, our results indicate that impairment of the interaction of filamin A with STIM1 induced by filamin A knockdown results in enhanced platelet aggregation in response to thrombin [FLNA gene, resulting in a 100-amino acid-long filamin A C-terminal extension, exhibits upregulated platelet aggregation and secretion in response to agonists. This filamin A mutation has been shown to lead to increased αIIbβ3 activation and the enhanced platelet function [SOCE in platelets requires a profound remodeling of the actin cytoskeleton, which acts as a negative clamp to prevent spontaneous SOCE activation but also is necessary to support the mechanism of Ca of SOCE . To illu of SOCE . Filaminth STIM1 . Impairmthrombin . These ffunction . Furtherfunction .2+ signals, is among the mechanisms underlying the modulatory role of filamin A in platelet function. This study raises several questions in the context of platelet function and the development of hemostatic and cardiovascular disorders. Among the most closely related are the possible involvement of filamin A in uncharacterized thrombotic or bleeding disorders as well as the possible regulation of filamin A expression associated with age.Summarizing, filamin A plays a relevant role in fine-tuning the platelet function in response to physiological agonists. The regulation of STIM1-Orai1 interaction, and, therefore, the extent of SOCE to mediate physiologically relevant Ca


Zebrafish and Drosophila models as well as the mammalian rodent systems, comprising rats and mice. The suitability of each animal model, its contribution to the advancement of knowledge in AML pathophysiology and treatment, as well as its advantages and limitations are discussed. Despite some limitations, animal models represent a powerful approach to assess toxicity, and permit the design of new therapeutic strategies.Acute myeloid leukemia (AML) is one of the most frequent, complex, and heterogeneous hematological malignancies. AML prognosis largely depends on acquired cytogenetic, epigenetic, and molecular abnormalities. Despite the improvement in understanding the biology of AML, survival rates remain quite low. Animal models offer a valuable tool to recapitulate different AML subtypes, and to assess the potential role of novel and known mutations in disease progression. This review provides a comprehensive and critical overview of select available AML animal models. These include the non-mammalian  Nucleophosmin-1 (NPM1), DNA methyltransferase 3A (DNMT3A), Fms-like tyrosine kinase-3 (FLT3), isocitrate dehydrogenase (IDH), Ten–Eleven Translocation 2 (TET-2), Runt-related transcription factor (RUNX-1), CCAAT enhancer binding protein α (CEBPA), additional sex comb-like 1 (ASXL1), mixed lineage leukemia (MLL), tumor protein p53 (TP53), c-KIT  model is closer to AML [−/Sca-1+/c-Kit+) populations. hBCL-2 is observed in the increased RAS-GTP complex within the expanded Sca-1+ compartment [BCL-2 and mutant N-RAS can cooperate to give rise to malignant disease with a penetrance of around 80% and a latency period of 3 to 6 months [Two murine models of initiation and progression of human MDS/AML were generated . The trar to AML . Both mopartment . The difpartment . Downregpartment . This mo6 months .MLL-PTD) is expressed in 5 to 7% of cytogenetically normal (CN)-AML patients [PTD/WT:flt3ITD/WTMll AML found in humans, a double knock-in mouse model was generated by expressing these two mutated genes under their respective endogenous promoters [MLL-WT expression, loss of FLT3-WT, and increased total FLT3 expression [HOXA9 transcript levels were observed, rendering this model valuable for the assessment of epigenetic modifying agents combined with tyrosine kinase inhibitors [Mixed lineage leukemia-partial tandem duplication , a structural component of the nuclear pore complex, to the homeobox protein NHD13 (HOXD13), inducing leukemogenesis [NUP98-HOX fusions are observed in human and murine MDS [NUP98-HOX correlated with higher transcript levels of FLT3 and an increased incidence of FLT3 activating mutations [NHD13 transgene enhanced the overexpression of the HOX genes, HOXA7, HOXA9, HOXB4, HOXB6, HOXB7, HOXC4, and HOXC6 [FLT3 allele, [The chromosomal translocation t leads to the fusion of Nucleoporin ogenesis . NUP98-Hrine MDS . Clinicarine MDS . High-leutations . A novelutations . Initialutations . Indeed,nd HOXC6 , shown tnd HOXC6 ,188,189. allele, , a chara allele, . These t allele, .flox−cA/+Npm1 with constitutive ITD/+Flt3 mice [flox−cANpm1 in hematopoietic stem cells was accomplished by crossing the double heterozygous mice into Mx1-Cre transgenic mice [NPM1c+ and FLT3-ITD double mutations are found in about 40% of AML patients . A compoD/+ mice . Inducinnic mice . Double nic mice . Solid onic mice .LSL-G12D; Cbfb56MNras) with an allelic expression of oncogenic N-RASG12D and CBFB-MYH11 developed leukemia in a cell-autonomous manner, with a short median latency and high leukemia-initiating cell activity [LSL-G12D; Cbfb56MNras leukemic cells were sensitive to pharmacologic inhibition of the MEK/ERK signaling pathway [G12D AML [A knock-in mice  developed high penetrance, enhanced self-renewal capacity in hematopoietic progenitors, and AML-like myeloid differentiation bias [N-RAS-G12D allele was observed, along with other somatic mutations in AML driver genes [HOX genes, which were overexpressed, HOXa genes and downstream targets were crucial for the survival of the double-mutant mice [One of the most common mutations with NPM1c+ is the patients . NPM1 anion bias . At the er genes . Within ant mice .WT1 occur in 10–15% of CN-AML, and it is frequently associated with mutations in several genes [FLT3-ITD and WT1 mutations, when present concomitantly, identify a group of AML patients that fail to respond to the standard induction chemotherapy, which results in poor OS [Flt3+/ITD/Wt1+/R394W displayed manifestations of shortened survival, myeloid expansion in the BM, anemia, and erythroid dysplasia [Wilms tumor 1 (WT1) is a zinc finger transcriptional regulator of target genes implicated in cell differentiation and quiescence . Mutatioal genes ,195. FLT poor OS . Double ysplasia . Althougysplasia .Humanized mouse models, injected with AML cell lines or patient-derived AML blasts, offered a faster approach and were instrumental in studying different aspects of AML. Several models were attempted to study AML in Nude mice with little success ,199. ThiSCID) mice lacking B and T cell immunity [+ CD38− surface markers, recapitulating the human HSCs signature [SCID mice reflected their intrinsic biologic behavior, suggesting a clinical correlation to the growth and dissemination of these leukemic subtypes [SCID mutation occurring in around 10% of the mice [The severe combined immuno-deficient  model exhibiting further impairment of NK activity, reduced mature macrophage, and total lack of B and T cells was generated [+ CD38−) into CD34+/CD71−/HLA-DR [SCID model presents the limitation by which higher engraftment rates required the supplementation of human cytokines or transplantation of growth-factor producing cells [SCID background is the NSS model (N/S-S/GM/3) expressing Steel factor (SF), granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) human growth factors was generated [To further improve tumor engraftment, a non-obese diabetic  translocation) leads to AML1-ETO degradation and impairs their clonogenic potential [The chromosomal translocation t(8:21) is frequent and common in AML. It represents up to 40% of AML subtype M2 of the FAB classification . The fus mammals . Two AMLo-factor . The traopoiesis . Using topoiesis . Osman eotential . Anotherotential . Thus, ADespite advances in our understanding of many molecular mechanisms, in vitro research falls short in determining overall effect of treatment modalities or drug discovery. AML is an intricate disease where culture consisting of a single cell line system, can never recapitulate the complexity of the disease. In the difficulty of obtaining primate models of AML, small rodents, zebrafish, and Drosophila with well characterized genetic background and relative ease of manipulation, are the backbone of current work where leukemic cells are interfaced with the host immunity, metabolic environment and importance of the niche ation. Not one model is sufficient to address all posed questions. However, collectively, these models have expanded our knowledge and understanding of several pathways and important players in AML pathogenesis.


Pancreas is one of the organs most frequently damaged by the disease leading to pancreatic insufficiency, abdominal pain and an increased risk of acute pancreatitis in CF patients causing a significant decrease in the quality of life. CFTR plays a central role in the pancreatic ductal secretory functions by carrying Cl- and HCO3- ions across the apical membrane. Therefore pathophysiological studies in CF mostly focused on the effects of impaired ion secretion by pancreatic ductal epithelial cells leading to exocrine pancreatic damage. However, several studies indicated that CFTR has a central role in the regulation of intracellular signaling processes and is now more widely considered as a signaling hub in epithelial cells. In contrast, elevated intracellular Ca2+ level was observed in the lack of functional CFTR in different cell types including airway epithelial cells. In addition, impaired CFTR expression has been correlated with damaged mitochondrial function in epithelial cells. These alterations of intracellular signaling in CF are not well characterized in the exocrine pancreas yet. Therefore in this review we would like to summarize the complex role of CFTR in the exocrine pancreas with a special focus on the intracellular signaling and mitochondrial function.Cystic fibrosis (CF) is the most common genetic disorder that causes a significant damage in secretory epithelial cells due to the defective ion flux across the cystic fibrosis transmembrane conductance regulator (CFTR) Cl This protein consists of five domains: two transmembrane domains, two cytoplasmic nucleotide-binding domains (NBD) and a regulatory (R) domain [interested readers can find detailed reviews about the structure and the structure- function relationships of CFTR in recent publications  – but not CFTR – on the apical membrane, which channel’s anion selectivity can be dynamically modulated by the Ca2+/calmodulin complex making it permeable for HCO3- (+/H+ exchanger regulatory factor-1 (NHERF1) knockout mice, due to the loss of CFTR leads to more severe experimental AP /STE20/SPS1-related proline/alanine-rich kinase (SPAK) kinase pathway switching CFTR permeability in favor of HCO3- . In a recent publication 3) receptor-binding protein released with InsP3 (IRBIT) controls the synergy between the Ca2+ and cAMP signaling pathways. The authors demonstrated that co-expression of CFTR and SLC26A6 with IRBIT maximally activate the two proteins in response to low agonist concentration, whereas IRBIT knockdown reduced their activation. PKA activation with forskolin left shifted the concentration-dependence of SLC26A6 activation by Ca2+ without directly activating SLC26A6. In contrast, Ca2+ signaling evoked by carbachol enhanced activation of CFTR by cAMP. The synergy was mediated by intracellular release of IP3 rather than Ca2+ since the synergism by IRBIT was independent of cytoplasmic Ca2+.Another well-known fact is that the increase of intracellular Caimulated . In a re2+ regulated AC1, AC5, AC6, and AC8 are targeted into lipid rafts in the plasma membrane, whereas the Ca2+ independent AC2 and AC7 are excluded from these rafts  (which inhibits Src tyrosine kinase) abolished the PKA-independent component of muscarinic stimulation. Based on their results the authors proposed that Ca2+ activates CFTR, at least in part, through activation of the Pyk2/Src complex and tyrosine phosphorylation. Another interesting aspect of PKA-independent activation of CFTR by Ca2+ elevation was recently reported by 2+-, and phosphorylation-dependent manner. This interaction triggered an open probability of CFTR, which was similar to that induced by PKA phosphorylation.2+ concentration is elevated in different cell types (fibroblasts and lymphocytes) both from homozygous or heterozygous carriers as demonstrated by atomic absorption spectrophotometry  and plasma membrane Ca2+-ATPase (PMCA) pumps can contribute to the deregulation of the Ca2+ homeostasis in CF. The activity of the SERCA pump and the ER Ca2+ concentration were strongly increased, while PMCA function is significantly impaired in CF bronchial epithelial cells. They also confirmed the earlier findings on the elevated mitochondrial Ca2+ uptake in CF cells compared to controls. Treatment with the CFTR folding corrector VX-809 reversed the alterations of the intracellular Ca2+ signaling. Interestingly, they demonstrated the physical interaction of SERCA2b with CFTR, and PMCA with CFTR. 2+ mobilization decreased compared to uncorrected cells. In another study the contribution of transient receptor potential canonical 6 (TRPC6) channel has been demonstrated to the increased intracellular Ca2+ levels. This study showed that CFTR and TRPC6 are functionally coupled within a molecular complex in airway epithelial cells, which is lost in CF leading to abnormally increased TRPC6-dependent Ca2+ influx  responsible for Ca2+ uptake (+/Ca2+ exchanger (NCLX)  opening that results in the loss of mitochondrial membrane potential, uncoupling of the respiratory chain and a consequent drop of mitochondrial ATP synthesis. The increased permeability of the inner mitochondrial membrane eventually leads to mitochondrial swelling, rupture, and necrotic cell death   for the ll death . The invroblasts  and incrroblasts . Similarncreased . The autncreased , which increased . As a coocessing . In accoocessing . As the ocessing , while oocessing . The altocessing . Other sP. aeruginosa, whereas 83% of all CF patients are in need of pancreatic enzyme replacement therapy causing a significant burden to the patients and to the healthcare systems.Cystic fibrosis is the most common autosomal recessive genetic disease characterized by multiorgan pathology and significantly decreased life expectancy caused by the impaired function or expression of CFTR. Due to recent therapeutic developments, the life expectancy of patients with CF elevated significantly in the past decades, however, the average age of death  is still 31.4 years . In addiCFTR gene mutations have been described, whereas only 159 mutations have been characterized in terms of disease liability  is the progressive inflammation of the pancreas accompanied by irreversible functional (exocrine and endocrine pancreatic insufficiency) and structural changes  . The inced in CP , in accoed in CP . On the ed in CP , which f2+ signaling and mitochondrial dysfunction. As detailed above both impaired regulation of the intracellular Ca2+ concentration and the mitochondrial damage develop due to complex mechanisms that can further ameliorate cell function and increase cellular damage complicating the organ damage in CF. These alterations have been described mainly in airway epithelial cells and we have very limited information how these mechanisms contribute to CF-related cell damage in other organs. However, taken into consideration that CFTR is highly expressed in the polarized pancreatic ductal cells and plays a pivotal role in the exocrine cell secretion, we could assume that these intracellular changes have a central role in the development of exocrine pancreatic damage in CF. In a recent case report 2+ elevation and mitochondrial damage are hallmarks of AP, restoration of CFTR activity, or expression could potentially decrease the cell damage in AP (In this review we emphasize the effect of CFTR on intracellular signaling events focusing on the intracellular Cage in AP . FurtherTM and PP collected, analyzed, and summarized the literature and drafted the paper. JM collected and analyzed the literature and drafted the manuscript.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.


Reverse-transcription PCR; western blotting; ELISA and luciferase assays were used to test the effect of compound 1 on EGF-stimulated expressions of COX-2 and IL-8 in A431 human epidermoid carcinoma cells. After exposure to 10 μM of compound 1, expression levels of COX-2 and IL-8 were reduced. In addition; intracellular Ca2+ increase and Ca2+-dependent transcription factor activation were blocked by compound 1. Thus, compound 1 can potentially serve as a lead compound for targeting Ca2+ signaling-dependent inflammatory diseases.Epidermal growth factor receptor (EGFR) is overexpressed in many types of cancer cells. EGFR-mediated signaling involves inflammatory gene expression including cyclooxygenase (COX)-2 and interleukin (IL)-8, and is associated with cancer pathogenesis. In a search of phytochemicals with anti-inflammatory activity, the COX-2 and IL-8 inhibitory activities of some marine compounds were examined. After screening these compounds 11-episinulariolide acetate ( Fluo-4 signals were calibrated by measuring the fluorescence intensity from microcuvettes containing 10 mM K2EGTA (pH 7.20) buffered to various [Ca2+] levels. The following formula was used for Ca2+ concentration calculation: [Ca2+]I = KD × (F − Fmin/Fmax − F). Plotting the fluorescence intensity versus [Ca2+] yielded the calibration curve with the formula:Cytoplasmic calcium signal was detected with stimulation by EGF or thapsigargin (TG) . Cells were incubated with 1 μM Fluo-4-AM  at 37 °C for 20 min. Then cells were washed with BSS buffer (5.4 mM KCl, 5.5 mM D-glucose, 1 mM MgSOr Fluo-4 .1 isolated from a soft coral. Compound 1 targeted EGF-mediated cytoplasmic calcium, which contributed to the inhibition of EGF-mediated COX-2 and IL-8 expressions. Ca2+ signaling has recently received greater attention in cancer research. Inhibition of store-operated Ca2+ influx may prevent the proliferation, metastasis, and angiogenesis of cancer cells [1 can potentially serve as a lead compound for targeting store-operated calcium signaling-dependent inflammatory diseases.We identified the anti-inflammatory effects of compound er cells ,29,30 (F


If is upregulated in failing and infarcted hearts, but its implication in disease mechanisms remained unresolved. We generated transgenic mice (HCN4tg/wt) to assess functional consequences of HCN4 overexpression-mediated If increase in cardiomyocytes to levels observed in human heart failure. HCN4tg/wt animals exhibit a dilated cardiomyopathy phenotype with increased cellular arrhythmogenicity but unchanged heart rate and conduction parameters. If augmentation induces a diastolic Na+ influx shifting the Na+/Ca2+ exchanger equilibrium towards ‘reverse mode’ leading to increased [Ca2+]i. Changed Ca2+ homeostasis results in significantly higher systolic [Ca2+]i transients and stimulates apoptosis. Pharmacological inhibition of If prevents the rise of [Ca2+]i and protects from ventricular remodeling. Here we report that augmented myocardial If alters intracellular Ca2+ homeostasis leading to structural cardiac changes and increased arrhythmogenicity. Inhibition of myocardial Ifper se may constitute a therapeutic mechanism to prevent cardiomyopathy.HCN channels underlie the depolarizing funny current (I The depolarizing funny current contributing to cardiac pacemaking is upregulated in the myocardium of  failing and infarcted hearts, but whether the current is implied in disease mechanisms is unclear. Here the authors generate HCN4 transgenic mice and show that upregulation of funny current to the levels observed in human heart failure alters calcium homeostasis leading to cardiac remodelling and arrhythmia. Apart from its abundant expression in the pacemaker and conduction system, the adult working myocardium is characterized by low HCN levels6. At early embryonic stages, however, HCN4 is abundantly transcribed in the whole heart6 and contributes importantly to If triggered automaticity of ventricular myocytes7. Moreover, HCN4 was identified as a cell marker for the cardiomyogenic progenitor pool of the first heart field, implicated in the earliest stage of cardiac mesoderm formation and morphogenesis10. Toward birth HCN4 transcription is downregulated in working-type cardiomyocytes6 and remains at low levels during adult stages, suggested to prevent pathological remodeling6. In this context, increased If and HCN expression was reported in failing hearts and after myocardial infarction12, but its pathophysiological role remains to be established. Notably, pharmacological blockade of If improves cardiovascular outcome in patients with chronic heart failure—results that were considered to be driven mainly by improved myocardial energy supply at lower heart rates14. However, recent data demonstrated that If blockade in patients with coronary artery disease with preserved myocardial function did not improve outcome despite marked reduction of heart rate15, pointing towards mechanisms of If blockade that may be particularly beneficial in failing hearts. In this regard, pleiotropic effects of If blockade beyond heart rate reduction have been suggested17, but these mechanisms have not been specified yet.The hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) is the dominant HCN-isoform in the sinoatrial node and is significantly involved in generation and regulation of heart rhythmf in the working myocardium, we generated transgenic mice that express human HCN4 (hHCN4) under control of the murine cardiac troponin I (cTNI) gene promoter. We here show that a moderate increase of If in cardiac myocytes to levels observed in heart failure leads to cardiac dilation and impaired cardiac function. HCN4 activity essentially is linked to calcium regulation of cardiomyocytes and augmented If results in dysregulated Ca2+ homeostasis driving cell death and cardiac remodeling.To study the significance of increased If augmentation in the working myocardium, transgenic mice were generated that express hHCN4 under control of the murine cTNI gene promoter19  of ventricular cardiomyocytes (~−90 mV) we observed a two–three3-fold higher If density in HCN4tg/wt cardiomyocytes compared to wild type    Fig. . Furtherst) Fig. .Fig. 2MoHCN4tg/wt hearts, we observed significantly increased diameters of cardiomyocytes , CnA  and NPPA (encoding ANP)  and KCND2 (encoding Kv4.2) were decreased, while the repolarizing channel gene KCNH2 (encoding Kv11.1) showed a trend toward higher levels  Fig. . We furtNP) Fig. . InteresCX1 Fig.  and intrhed Fig. . Furtherels Fig. . Other gNCX1 downregulation in HCN4tg/wt hearts, we hypothesized that dysregulated intracellular sodium–calcium homeostasis might be a pathomechanistic component in If overexpression-mediated cardiomyopathy. To evaluate a contribution of calcium handling proteins we performed immunoblotting of various key players. In line with the transcriptional analysis we observed significantly reduced levels of NCX1 protein in HCN4tg/wt hearts, a finding that was paralleled by lower levels of SERCA2 and phospholamban (PLN) whole protein content  of calcium transients  in 39% of cardiomyocytes isolated from HCN4tg/wt hearts  of calcium transients  reversed the changes in Caels Fig. . Increasbly Fig.  and the els Fig. . Moreoveake Fig.  and shornts Fig.  were res2+]i would provoke apoptosis in HCN4tg/wt hearts. Using TUNEL assay, we detected significantly higher proportions of nuclei with DNA fragmentation in ventricles of HCN4tg/wt hearts compared to wild type    and 50% (APD50) repolarization were shorter in transgenic cells, with significant changes of APD50 at all pacing frequencies and of APD20 at 0.5 Hz. Evaluation of APD90, by contrast, did not differ significantly between wild type and HCN4tg/wt cardiomyocytes  recordings of que Fig. . In HCN4st) Fig. , consistst) Fig. , pointinHz) Fig. . AP durates Fig. .Fig. 8Elf and consecutive Ca2+ overload we asked whether HCN4tg/wt cardiomyocytes elicit increased cellular automaticity and/or arrhythmogenesis. Recordings of [Ca2+]i transients and APs  than in wild type cardiomyocytes  . Remarkably, iva treatment diminished spontaneous firing and ADs, and percentage of HCN4tg/wt cardiomyocytes showing arrhythmic behavior after treatment was not different from wild type  Fig. . To deliype Fig. , demonstHCN4tg/wtmice we performed ECG recordings under anesthesia and telemetric recordings in freely roaming animals. Both approaches revealed similar heart rates and conduction parameters among transgenic (HCN4tg/wt) and wild type mice and did not exhibit significant changes of additional ECG parameters  under control of the murine cTNI promoter. While abundantly expressed in the working myocardium, cTNI is downregulated in the SAN and the conduction system22. This is phenotypically reflected by telemetric recordings revealing similar heart rates and conduction parameters in transgenic and wild type mice  patch clamp recordings revealed a two–three-fold higher If density in HCN4tg/wt cardiomyocytes compared to wild type 31 and the close interrelation of intracellular Na+ and Ca2+ homeostasis32, we asked how increased If might influence Ca2+ cycling. To elucidate the impact of increased If, we measured [Ca2+]i transients in isolated, electrically driven cardiomyocytes in the pre- and absence of ivabradine. Strikingly, we observed a marked rise in [Ca2+]i baseline levels and significantly augmented systolic [Ca2+]i transients in transgenic cardiomyocytes – changes that were fully recovered in the presence of ivabradine. Thus, in the myocardium chronic upregulation of If exerts a diastolic Ca2+ overload and importantly interferes with cellular Ca2+ homeostasis.Given the primary Na2+ efflux in cardiac myocytes is via electrogenic Na+/Ca2+ -exchange (NCX), Ca2+ homeostasis is tightly linked to Na+ regulation32. Under physiological conditions, NCX primarily operates in (forward) Na+ -in/ Ca2+ -out mode, and reduces diastolic [Ca2+]i levels by extruding Ca2+. In pathological states, however, when [Na+]i is increased, NCX exerts ‘reverse mode’ function, moving Ca2+ into the cell32. Notably, [Na+]i is known to rise in a rate dependent fashion32, which exacerbates [Ca2+]i overload at high heart rates. The impact of If on [Na+]i, in relation to membrane potential has been demonstrated in sheep Purkinje fibers by voltage-clamp recordings in early works34 after the If mixed Na+ and K+ ionic nature was originally described35. These studies showed that membrane hyperpolarization to below −60 mV significantly increased intracellular Na+ activity, and related this increase to Na+ influx through If, thus providing a direct link between [Na+]i and the size of If. Based on these data, we hypothesized that HCN4 overexpression-mediated accumulation of [Na+]i may raise Ca2+ influx via reverse mode NCX activity , while inward tail current of If prolonged APD at membrane potentials more negative than reversal potential. However, prolongation of APD90 was not observed in our model, most likely due to the repolarizing driving force of the electrogenic ‘reverse mode’ NCX, known to effectively shorten AP duration when [Na+]i is increased39.We next addressed the effects of increased Itg/wt hearts showed a higher incidence of spontaneous APs than wild type cells, and a subset of cells exerted virtual pacemaker function. The increased automaticity may originate from If augmentation and not from spontaneous Ca2+ cycling-mediated membrane depolarisation, as the NCX equilibrium is shifted toward ‘reverse mode’ leading to an outward, hyperpolarizing current. Moreover, HCN4tg/wt cardiomyocytes were prone to afterdepolarizations resulting in trains of premature APs. Recording of calcium transients showed abnormal diastolic Ca2+ decay in 39% of HCN4tg/wt cells, produced by SR Ca2+ overload, promoting afterdepolarizations and arrhythmic firing40. However, during telemetric recordings HCN4tg/wt animals exhibited no sustained arrhythmias, although higher numbers of PVCs and few nonsustained VTs indicated increased arrhythmogenicity compared to wild type animals. This imbalance between cellular arrhythmogenicity and lack of high-grade arrhythmias in vivo might be explained by source-sink mismatch41 proposing that propagation of ADs originating from single cells (source) is hindered by the rectifying potassium conductance of neighboring myocytes within the myocardial syncytium (sink), thereby protecting the heart from deleterious arrhythmias.Interestingly, we found that myocytes isolated from HCN444. Harmful [Ca2+]i promotes cardiomyocyte death through apoptotic or necrotic pathways43. Previous studies have delineated the association between apoptotic cell-loss and progression of heart failure in animal models and in humans46. Moreover, Ca2+ overload induced by persistently activated L-type calcium channels47 or chronically compromised Ca2+ removal systems47 were shown to induce myocardial cell death and remodeling. Accordingly, the structural changes in our model can be explained by the observed caspase-3-mediated apoptosis. Likewise, we found higher transcript levels of tissue transglutaminase and the Ca2+-sensor protease μ-calpain  by pronuclear injection of an 8.2 kb DNA fragment carrying a 4.3 kb promoter fragment of the murine cTnI gene19 joined to the 5′ end of the human HCN4 cDNA48 that was fused to the bovine growth hormone gene poly A signal . All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health , and with the European Community guidelines for the use of experimental animals as well as all relevant ethical regulations. Protocols were approved by the local regulatory authority . We used male mice for all experiments.Mixed genotype groups of each gender of no more than five animals were housed in standard mice were anesthetized with isoflurane vapor titration to maintain the lightest anesthesia possible49. On average, 1.5% vol/vol isoflurane was required to maintain adequate anesthesia. Mice were placed on a heating pad with continuous monitoring of body temperature  maintaining at 37 °C. Surface ECGs were recorded using clamp electrodes attached to each limb and to the chest in a midsternal position. The electrodes were connected to a Powerlab System  and ECG recording were performed with a high and low frequency cut off of 1 kHz and 0,1 kHz. ECG recordings were analysed by an investigator blinded to the experimental groups using the Chart 5 software . In addition to average heart rate, at least 30 complexes of each trace were signal averaged and the following parameters were evaluated in lead II: PR-interval, QRS complex duration, and QT-interval. Corrected QT intervals were calculated according to Mitchell et al., 199850, QTc = QT/(RR/100)1/2.For surface ECG recordings Mice were anesthetized intraperitoneally with a mixture of ketamine (100 mg/kg) and xylazine (3 mg/kg). The leads were tunnelled under the skin to the recording sites, i.e. one subcutaneous lead placed at the right shoulder and a second subcutaneous lead placed at the lower left thorax. The abdominal skin was closed and mice were kept on a heating pad (37–38 °C) until they were fully recovered from anaesthesia. Before measurement, mice were allowed to recover for 1 week, followed by 24 h recording. ECG signals were converted to digital output and stored on a computer using the Powerlab System , analysis was carried out with the Chart 5 software .For telemetric ECG recordings, a radiotelemetry transmitter  was surgically inserted into the peritoneal cavity. Mice were shaved, and left ventricular parasternal short-axis views were obtained in M-mode imaging at the papillary muscle level. Three consecutive beats were used for measurements of left ventricular end-diastolic internal diameter (LVEDD) and left ventricular end-systolic internal diameter (LVESD). Fractional shortening (FS) was calculated as FS% = ((LVEDD − LVESD)/LVEDD) × 100.The animals were sedated with low-dose isoflurane  and placed on a heating pad in supine position. A rectal probe was inserted to monitor the body temperature to maintain it at 37 °C. Chest hair was carefully removed with depilatory cream. Warmed echo gel was placed on the animal’s chest. The Vevo® 2100 device  with the corresponding transducer MS-400 was used to record the echocardiographies. The investigator was blinded with respect to the treatment group. mice. Hearts were excised, ventricular tissue was carefully dissected and immediately frozen in liquid nitrogen. Total RNA was extracted using TRIzol-Reagent®  according to the manufacturer’s instructions, followed by reverse transcription . Quantitative real-time PCR (RT-qPCR) was performed using an ABS 7500 Realtime PCR System  according to the manufacturer’s protocol. 96 well optical detection plates  were loaded to a total volume of 10 µl per well, consisting of 0.5 µl cDNA, 5 µl TaqMan Fast Universal Master Mix , and 6-carboxyfluorescein (FAM)-labeled pre-designed TaqMan primers  specific to human and mouse HCN4 transcripts, HCN1, HCN2. qRT-PCR of apoptosis and Ca2+ processing related genes was performed using TaqMan assays for Casp3, tTG, Capn1, RyR2, Serca2a, Pln, Na+/K+-ATPase and NCX1. Ion channel transcripts were examined using TaqMan assays for SCN5A, Cav1.2, Cav1.3, Cav2.1, Cav3.1, Kir2.1, Kv1.4, Kv1.5, Kv4.2, Kv7.1, hERG, and KChip2. Transcripts of myocardial cell growth marker genes and genes of the fetal gene program were determined with TaqMan assays for mTOR, GSK-3B, ANP, Calcineurin and β-MHC. Predesigned primers and probes detecting the housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), and beta-actin (ACTB) were applied and normalization was carried out by a modified threshold cycle (CT) relative quantification method using GAPDH  on the manufacturer’s website http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php.Expression of apoptosis related genes was screened with Mouse Apoptosis RT² Profiler PCR Array  on an ABS 7500 Realtime PCR System and data was analysed using online software “RTmouse left and right ventricle samples were rinsed in phosphate buffered saline (PBS), rapidly frozen in liquid nitrogen and stored at −80 °C. Tissue samples were homogenized  in a radioimmunoprecipitation (RIPA) lysis buffer containing 50 mM Tris-HCl (pH 7.4), 0.5% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, and protease inhibitors . The protein concentration was determined using the bicinchoninic acid (BCA) protein assay , and equal amounts of protein were separated on SDS polyacrylamide gels. Nitrocellulose membranes were developed by sequential exposure to blocking reagent containing 3% bovine serum albumin and 5% dry milk. Primary antibodies were directed against HCN4 (1:200 diluted in 5% dry milk), NCX1 (1:250), Cav1.2 (1:500), Serca2 (1:1000), Phospholamban (1:500), phosphorylated Phospholamban Thr-17 (1:2000), phosphorylated Phospholamban Ser-16 (1:2000) and Calpain1 (1:2000). Appropriate HRP-conjugated secondary antibodies  were used. Signals were developed using an enhanced chemiluminescence assay  and quantified with ImageJ 1.41 Software . Protein content was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using anti-GAPDH primary antibodies  and corresponding secondary antibodies for digital quantification of optical density. For detailed information on primary and secondary antibodies used please refer to Supplementary Table Protein immunodetection was performed by sodium dodecyl sulfate (SDS) gel electrophoresis. Tissue sections obtained from indicated 52. Briefly, the mice received 200 IU heparin i.p. prior to sacrifice. The thoracic chamber was opened and a cannula with perfusion solution was inserted from the atria. The heart was harvested and perfused in the Langendorff system with Perfusion Buffer , followed by Digestion Buffer treatment , 0.4 mg/g body weight collagenase B , 0.05 mg/g body weight protease XIV  in 25 ml Perfusion Buffer) until the heart muscle was pale and some signs of extracellular matrix dissociation and for additional 5 min with Perfusion Buffer to stop the dissociation. The cardiomyocytes were mechanically dissociated in Transfer Buffer A in mM: 135 NaCl, 4 KCl, 1 MgCl2, 10 HEPES, 0.33 NaH2PO4, 5.5 glucose, 10 2,3-butanediones monoxime, 5 mg/ml bovine serum albumin , pH 7.4 at 37 °C) and then plated on ECM-coated  petri dish . The extracellular calcium concentration was increased gradually in three consecutive steps from 0 mM (in Transfer Buffer A) to 0.06, 0.24 and 1.2 mM every 5 min. The cells where then incubated at 37 °C in 5% CO2.Cardiomyocytes for intracellular calcium measurements were obtained following the Liao & Jain protocolf was recorded under voltage-clamp conditions (whole-cell configuration) in isolated mouse ventricular cardiomyocytes at room temperature (21–23 °C), essentially as published54. In detail, patch-clamp measurements of mouse ventricular cardiomyocytes were performed in a solution containing (in mM): 137 NaCl, 25 KCl, 8 BaCl, 2 CaCl2, 2 MnCl, 0.2 CdCl, 0.5 4-Aminopyridine, 5 HEPES, 10 Glucose, pH corrected to 7.4 using 1 M NaOH. The patch pipettes pulled from borosilicate glass  were generated on a DMZ Universal puller  and fire polished to give a final resistance of 2.5–4 MΩ. The pipette solution contained (in mM): 130 K-Aspartate, 5 Na2ATP, 5 CaCl2, 2 MgCl2, 11 EGTA and 10 HEPES, pH corrected to 7.3 using 1 M KOH. Pharmacological agents were added to the bath solution to block L- type calcium currents (10 μM nisoldipine), the slow component of delayed rectifier K+ current , the rapid component of delayed rectifier K+ current , ATP-dependent K+ current  and inward rectifier K+ currents . If currents were normalized to cell capacitance which was obtained from the time constant of a current transient evoked by a 5 mV potential step at the beginning of each sweep.I2, 0.5 MgCl2, 10 HEPES, 5.5 glucose, and a pipiette solution containing (in mM): 120 K-Aspartate, 10 KCl, 5 NaCl, 2 MgCl2, 10 HEPES. For quantification of AP duration and amplitude, cells were held in current-clamp mode and current was injected to achieve a membrane potential of −90 mV. If injected currents exceeded 200pA, recordings were discarded and not included in analysis. APs were evoked by 5 ms suprathreshold depolarizing current injection at 0.5, 1, and 2 Hz frequencies. Quantification of resting membrane potential, and recordings of spontaneous APs and afterdepolarizations were performed without current injection.Recordings of cardiac action potentials were carried out in an extracellular solution containing (in mM): 137 NaCl, 5.4 KCl, 1.8 CaClf and AP recordings were done with an Axopatch 200B amplifier , digitized at 20 kHz with a 1401 Power3 Analog/Digital Converter  stored on a hard drive, and analysed off-line with custom MATLAB routines  software. Recordings with less than 10% leak current were considered for data analysis. No leak subtraction was performed during the experiments. Recordings were discarded if access resistance exceeded 20 MOhm or changed for more than 20% during the recording period.I2, 0.5 MgCl2, 10 HEPES, 5.5 glucose, pH 7.4 at 37 °C) for 30 min. For pharmacological treatments ivabradine  or ORM-10103  were dissolved in DMSO for stock solution. For working solution, both ivabradine or ORM-10103 were dissolved in Transfer Buffer B at 3 μM or at 10 µM, respectively, and incubated for 10 min prior to the Ca2+ recordings.The cardiomyocytes were loaded with 10 μM Fluo-4, AM  dissolved in Transfer Buffer B  or abnormal diastolic [Ca2+]i decay (defined as D90 > 2000ms) were excluded from regular analysis and evaluated in a separate record  to obtain Fmax values followed by application of zero-Ca2+ solution  to provide Fmin values. The Ca2+ signal (F) was then converted into [Ca2+]i according to Grynkiewicz et al. 198556.Calibration was performed at the end of each experiment in freshly loaded cells with Fluo-4, AM to transform voltage values into [Ca2+] = Kd(F-Fmin)/(Fmax-F), with Kd: apparent Ca2+ dissociation constant of the Fluo-4 of 345 nM, according to the manufacturer’s information.. The calculation was based on averaged measurements from 10 sections per animal . Bright-field images were digitally recorded on Axioplan2 microscope  with attached 3CCD camera .TUNEL assay was performed and quantified according to manufacturer’s protocol . Confocal images of red and green immunofluorescence staining of heart tissue sections were acquired on a Leica confocal laser scanning unit TCS NT, which was coupled to a Leica DM IRB microscope. Acquisition of image series was performed using TCS NT  software. All images were processed in ImageJ version 1.47  software using standard quantification methods and standard ImageJ plug-ins.tg/wt and wild type mice was dissected, and prepared for microscopy57. Briefly, cardiac tissue was fixed overnight at 4 °C in a calcium-free buffer . After several washes with 100 mM PIPES, pH 7.0, samples were incubated for 2 h at room temperature in aqueous 2% osmium tetroxide and 1.5% potassium-hexacyanoferrate solution, washed in water for 3 h and incubated in water with 4% uranyl acetate overnight at 4 °C. Samples were dehydrated as described57 and embedded in Epon medium. Ultra-sections were contrasted in uranyl acetate for up to 20 min and in lead citrate for 5 min. Sections were viewed on Philips EM400T  and images were documented on Image Plates and scanned with the Micro Imaging Plate Scanner .Right and left ventricular wall tissue of HCN4t-test. Comparisons between multiple groups were performed using one-way ANOVA followed by Bonferroni , Tukey  or Dunn’s  post-hoc tests. Differences were considered significant at a level P < 0.05. Data are presented as arithmetic mean ± s.e.m. or median ± maximum/minimum.All experiments and primary analyses were blinded. Statistical analysis was performed using GraphPad Prism 5.0 . Statistical significance and comparisons between samples were analyzed by a two-tailed unpaired Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource data


The significance of the osmosensitive [Ca2+]i signaling for renal water transport and urinary concentration remain unknown. To examine molecular mechanism and physiological relevance of osmosensitivity in the CD, we implemented simultaneous direct measurements of [Ca2+]i dynamics and the rate of cell swelling as a readout of the AQP2-dependent water reabsorption in freshly isolated split-opened CDs of wild type and genetically manipulated animals and combined this with immunofluorescent detection of AVP-induced AQP2 trafficking and assessment of systemic water balance. We identified the critical role of the Ca2+-permeable TRPC3 channel in osmosensitivity and water permeability in the CD. We further demonstrated that TRPC3 -/- mice exhibit impaired urinary concentration, larger urinary volume and a greater weight loss in response to water deprivation despite increased AVP levels and AQP2 abundance. TRPC3 deletion interfered with AQP2 translocation to the plasma membrane in response to water deprivation. In summary, we provide compelling multicomponent evidence in support of a critical contribution of TRPC3 in the CD for osmosensitivity and renal water handling.It is well-established that the kidney collecting duct (CD) plays a central role in regulation of systemic water homeostasis. Aquaporin 2 (AQP2)-dependent water reabsorption in the CD critically depends on the arginine vasopressin (AVP) antidiuretic input and the presence of a favorable osmotic gradient at the apical plasma membrane with tubular lumen being hypotonic compared to the cytosol. This osmotic difference creates a mechanical force leading to an increase in [Ca Kidneys play a central role in systemic water balance by excreting urine with a highly variable osmolarity depending on hydration status , 2. Anti2+]i [2+ entry from the extracellular medium leading to the elevation of [Ca2+]i to initiate cellular responses [2+]i elevations i . It is a2+]i , 13. Tra2+]i . TRP chaesponses , 15. Expesponses , 17. Accevations . Howeverevations , 21 indi2+]i plays an important role in synthesis and trafficking of AQP2 in the CD cells [2+]i signaling and whether it contributes to the regulation of the osmotically-driven water reabsorption in the CD cells.It has been reported that, in addition to cAMP, increased i signaling and water permeability and established an important role of the TRPC3 channel in both processes. We further uncovered that TRPC3 deficiency distorts subcellular AQP2 localization at the baseline and during antidiuresis and causes an impaired urinary concentrating ability and notable disturbances of systemic water balance, which cannot be compensated by high AVP levels. We further speculate that TRPC3-based pharmacological intervention might be of use to regulate urine production and osmolarity.In this study, we employed freshly isolated CDs to demonstrate a functional coupling between osmosensitive i and cell volume measurements closely followed the protocols previously published by our group for patch clamping and intracellular Ca2+ imaging i measurements, the Fura 2 fluorescence intensity ratio was determined by excitation at 340 nm and 380 nm and calculating the ratio of the emission intensities at 511 nm in the usual manner every 5 seconds. The changes in the ratio are were converted into changes in intracellular calcium, as documented previously i responses, the peak was calculated as the difference of the highest intracellular calcium concentration and the respective baseline.Intracellular calcium levels were continuously monitored in individual cells within a split-opened area of freshly isolated CDs using Fura 2 fluorescence imaging as described previously –34. Brieeviously . For monic point , were deic point . In averImmediately after dissection kidneys were placed on ice, decapsulated and homogenized in 5 volumes of hypotonic lysis buffer: 50 mM TrisCl, 5 mM EDTA, 1% Triton X-100 (pH = 7.5) supplemented with Complete Mini protease and PhosSTOP phosphatase inhibitor cocktails . The homogenates were centrifuged at 1000 g for 15 min at +4 ºC and the sediment was discarded. Protein concentration was determined with a Bradford assay using bovine serum albumin as a standard. The samples (20 μg/lane) were separated on 10% polyacrylamide gels at 150 V for 100 min and transferred to a nitrocellulose membrane for 110 min at 100 V. Subsequently the nitrocellulose membrane was incubated with primary antibodies for 2 hours at room temperature. The primary antibodies were anti-AQP2 , anti-AQP4  and anti-β-actin . Upon washout (3 times for 10 min in TBS-Tween) the membrane was incubated with peroxidase-conjugated goat anti-rabbit  secondary antibodies for 1 hour 30 min at room temperature. For AQP4, membranes were stained with Ponceau red (0.1% in 5% acetic acid) for 60 min. Blots were quantified using ImageJ 1.50 software . The intensities of the studied protein bands were normalized to the intensities of the corresponding β-actin bands used as a loading control or overall Ponceau signal in a line.2 camera (Photometrics) interfaced to a PC running NIS elements software.Kidneys were fixed in 10% neutral buffer formalin for 24 hours at +4°C and soaked in 0.9 M sucrose buffer for 4 hours for cryopreservation. Kidneys were further placed in O.C.T. Compound , snap frozen on dry ice and placed into -80°C. Transverse cut 6 μm thick sections were made on CM 1850 cryostat . The sections were allowed to warm to room temperature, washed 3 times in phosphate buffer saline (PBS), permeabilized with 1% SDS for 10 minutes following washout with PBS and blockade of non-specific binding with 1% BSA  for an hour at room temperature. Sections were further incubated overnight with anti-AQP2  followed by secondary antibodies Alexa 488  or Alexa 594 . Nuclei were stained with DAPI  for 10 minutes at room temperature. The labeled kidney sections were mounted with Fluoromount-G mounting media  and imaged with a Nikon A1R confocal microscope, as we did similarly before , 25. In All summarized data are reported as mean ± SEM. Statistical comparisons were made using one-way ANOVA with post-hoc Tukey test or one-way repeated measures ANOVA with post hoc Bonferroni test (for paired experiments within the same group). P value less than 0.05 was considered significant.2+]i. We and others demonstrated that the mechanosensitive Ca2+-permeable TRPV4 channel is essential for elevations in [Ca2+]i in response to high flow over the apical plasma membrane in the CD cells [2+]i by the same pathway. Thus, we first monitored changes in [Ca2+]i in individual cells in freshly isolated split-opened CDs in response to a shear stress of approximately 3 dyn/cm2  and appesponses . Howeveresponses . To acco-/- mice . In conttonicity . Altoget2+ may serve as a source of [Ca2+]i response to hypotonicity in CD cells. Blockade of the endoplasmic reticulum Ca2+ pump SERCA with thapsigargin (2 μM for 10 min) exerted only a mild (~20%), whilst significant, inhibitory effect on [Ca2+]i responses to application of hypotonic (220 mOsm) medium (2+ (5 mM EGTA) precluded elevations in [Ca2+]i induced by hypotonicity  medium . In conttonicity . We conc2+-permeable channels, including TRPC3 and TRPC6, are known to be expressed in the CD cells [2+]i elevations in response to application of 220 mOsm medium  and relative changes of cell volume (by quantifying changes in Fura-2 intensity with excitation at 360 nm (F360), the Ca2+-independent “isosbestic” point [2+]i and cell volume. As shown in 2+ (5 mM EGTA) markedly reduced [Ca2+]i levels (black), but did not affect the F360 signal (grey), suggesting no volume changes under this condition, as expected. Furthermore, application of hypertonic medium (400 mOsm) induced a reversible increase of F360 . Thus, 2+ entry is a critical determinant of [Ca2+]i responses to hypotonicity. Thus, we next investigated the relation between osmosensitive [Ca2+]i signaling and cell volume in the CDs from WT and TRPC3 -/- mice. 2+]i levels and relative changes in individual cell volume in the control, during application of a hypotonic medium (220 mOsm) for 10 min, and following washout with 300 mOsm in a CD from a WT mouse. The average time courses of [Ca2+]i and relative volume changes are shown in 2+]i response to hypotonicity constituted of a transient Ca2+ peak followed by a sustained plateau; and the respective cell volume response consisted of a rapid swelling phase followed by a slow volume recovery (i.e. a regulatory volume decrease). In CDs from TRPC3 -/- mice, the [Ca2+]i response to hypotonicity was nearly abolished , whereas the magnitude of the response is determined by the existing osmotic gradient [2+ (5 mM EGTA for 3 min) decreased basal [Ca2+]i levels and abolished [Ca2+]i response to hypotonicity in CDs from WT mice  stimulate the activity of another Ca2+-permeable channel, TRPC3. This argues that the CD cells are able to discriminate between specific types of mechanical stress on the plasma membrane by triggering a proper Ca2+-dependent response. Consistently, it was shown that fluid shear stress and circumferential stretch differentially affect ERK and p38 activation and PGE2 release in cortical CD cells [2+]i elevations. Indeed, while TRPV4 -/- mice demonstrate impaired sensitivity to renal tubular flow and have defective flow-induced K+ secretion in the CD [2+]i in CD cells. We can speculate that this is dictated by drastically different physiological conditions leading to activation of a respective channel. Thus, TRPV4-dependent Ca2+ influx would not increase apical water permeability, since AVP levels are low under conditions associated with increased tubular flow, such as high K+ diet. Similarly, TRPC3-dependent Ca2+ influx would not notably stimulate K+ secretion during antidiuresis, since tubular flow is not elevated as necessary for kaliuresis. It has to be noted though, that inhibition of Ca2+ release from the endoplasmic reticulum with thapsigargin also has a mild but significant effect on the magnitude of osmosensitive [Ca2+]i responses  mechanism, as was similarly reported for TRP channels [2+ abolished osmosensitive [Ca2+]i responses in CD cells  similarly in the center and periphery , a neurodevelopmental disorder commonly associated with hypercalcemia of unknown origin, has been recently linked to upregulation of TRPC3 expression due to deletion of the gene encoding the transcription factor TFII-I [2+ homeostasis [2+ handling.The major finding of this study is that activation of TRPC3 by luminal hypotonicity plays an important role in AQP2 trafficking to the apical plasma membrane. Augmented TRPC3-mediated [Catonicity . On the tonicity  or ablat WT mice  result i WT mice . This corivation . It shoution  and respective Ca2+ -independent Fura 2 fluorescence F360 (lower panel) in a split-opened CD from WT (A) and TRPC3 -/- (B) mice during perfusion with control 300 mOsm medium (left row), upon application of hypotonic 220 mOsm medium (middle row), and followed washout with the control medium (right row).Representative pseudocolor [Ca(TIFF)Click here for additional data file.S2 Fig(A) and TRPC3 (B) mice in the control (top) and after 24 hour water deprivation (bottom) used to visualize subcellular AQP2 localization, as shown in Low magnification images of transverse cut kidney sections from WT (TIFF)Click here for additional data file.S3 Fig2+ levels in the baseline and after 12h water deprivation in WT and TRPC3 -/- mice. Urinary Ca2+ levels were normalized on the respective osmolarities. *—significant decrease versus WT basal.Summary graph of spot urinary Ca(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.