MicroRNAs (miRs) contribute to different aspects of cardiovascular pathology, among others cardiac hypertrophy and atrial fibrillation. The aim of our study was to evaluate the impact of miR-221/222 on cardiac electrical remodeling. Cardiac miR expression was analyzed in a mouse model with altered electrocardiography parameters and severe heart hypertrophy. Next generation sequencing revealed 14 differentially expressed miRs in hypertrophic hearts, with miR-221 and-222 being the strongest regulated miR-cluster. This increase was restricted to cardiomyocytes and not observed in cardiac fibroblasts. Additionally, we evaluated the change of miR-221/222 in vivo in two models of pharmacologically induced heart hypertrophy (angiotensin II, isoprenaline), thereby demonstrating a stimulus-induced increase in miR-221/222 in vivo by angiotensin II but not by isoprenaline. Whole transcriptome analysis by RNA-seq and qRT-PCR validation revealed an enriched number of downregulated mRNAs coding for proteins located in the T-tubule, which are also predicted targets for miR-221/222. Among those, mRNAs were the L-type Ca 2+ channel subunits as well as potassium channel subunits. We confirmed that both miRs target the 3′-untranslated regions of Cacna1c and Kcnj5. Furthermore, enhanced expression of these miRs reduced L-type Ca 2+ channel and Kcnj5 channel abundance and function, which was analyzed by whole-cell patch clamp recordings or Western blot and flux measurements, respectively. miR-221 and-222 contribute to the regulation of L-type Ca 2+ channels as well as Kcnj5 channels and, therefore, potentially contribute to disturbed cardiac excitation generation and propagation. Future studies will have to evaluate the pathophysiological and clinical relevance of aberrant miR-221/222 expression for electrical remodeling. Keywords Electrical remodeling • Cardiomyocytes • Angiotensin II • Heart hypertrophy Abbreviations ANOVA Analysis of variance AUC Area under the curve AII Angiotensin II Bp Base pair BW Body weight Cacna1C Calcium voltage-gated channel subunit α1 C, L-type Cacnb2 Calcium voltage-gated channel auxiliary subunit β2 Cacna2d1 Voltage-dependent calcium channel subunit α2/δ1 Cav1.2 L-type Ca 2+ channel CCH Carbachol, acetylcholine analog cDNA Complementary DNA CE Carbachol effect CM Cardiomyocyte ddPCR Droplet digital PCR Cellular and Molecular Life Sciences
Background Cardiovascular disease is the leading cause of death worldwide. Cardiac electrical remodeling including altered ion channel expression and imbalance of calcium homeostasis can have detrimental effects on cardiac function. While it has been extensively reported that miR-221/222 are involved in structural remodeling, their role in electrical remodeling still has to be evaluated. We previously reported that subunits of the L-type Ca2+ channel (LTCC) are direct targets of miR-221/222. Furthermore, HL-1 cells transfected with miR-221 or -222 mimics showed a reduction in LTCC current density while the voltage-dependence of activation was not altered. The aim of the present study was to determine the influence of miR-221/222 on cardiomyocyte calcium handling and function. Results Transient transfection of HL-1 cells with miR-221/222 mimics led to slower depolarization-dependent Ca2+ entry and increased proportion of non-responding cells. Angiotensin II-induced Ca2+ release from the SR was not affected by miR-221/222. In miR-222-transfected neonatal cardiomyocytes the isoprenaline-induced positive inotropic effect on the intracellular Ca2+ transient was lost and the positive chronotropic effect on spontaneous beating activity was strongly reduced. This could have severe consequences for cardiomyocytes and could lead to a reduced contractility and systolic dysfunction of the whole heart. Conclusions This study adds a new role of miR-221/222 in cardiomyocytes by showing the impact on β-adrenergic regulation of LTCC function, calcium handling and beating frequency. Together with the previous report that miR-221/222 reduce GIRK1/4 function and LTCC current density, it expands our knowledge about the role of these miRs on cardiac ion channel regulation.
MicroRNAs (miRs) contribute to different aspects of cardiovascular pathology, among them cardiac hypertrophy and atrial fibrillation. Cardiac miR expression was analyzed in a mouse model with structural and electrical remodeling. Next-generation sequencing revealed that miR-208b-3p was ~25-fold upregulated. Therefore, the aim of our study was to evaluate the impact of miR-208b on cardiac protein expression. First, an undirected approach comparing whole RNA sequencing data to miR-walk 2.0 miR-208b 3′-UTR targets revealed 58 potential targets of miR-208b being regulated. We were able to show that miR-208b mimics bind to the 3′ untranslated region (UTR) of voltage-gated calcium channel subunit alpha1 C and Kcnj5, two predicted targets of miR-208b. Additionally, we demonstrated that miR-208b mimics reduce GIRK1/4 channel-dependent thallium ion flux in HL-1 cells. In a second undirected approach we performed mass spectrometry to identify the potential targets of miR-208b. We identified 40 potential targets by comparison to miR-walk 2.0 3′-UTR, 5′-UTR and CDS targets. Among those targets, Rock2 and Ran were upregulated in Western blots of HL-1 cells by miR-208b mimics. In summary, miR-208b targets the mRNAs of proteins involved in the generation of cardiac excitation and propagation, as well as of proteins involved in RNA translocation (Ran) and cardiac hypertrophic response (Rock2).
Introduction: Despite intensive research most patients with acute myeloid leukemia (AML) still have a dismal prognosis. The transcription factor RUNX1 is a master regulator of myeloid differentiation and in AML its function is often disrupted by chromosomal translocations or mutations. These genetic alterations impact on leukemogenesis, disease progression and prognosis of AML patients. The expression of fusion proteins involving RUNX1 has been shown to exert a dominant-negative effect over wild-type RUNX1 leading to impaired myeloid differentiation and enhanced proliferation, while loss of RUNX1 may impact negatively on the frequency and self-renewal capacities of long-term hematopoietic stem cells. However, it remains unclear if differential expression of RUNX1 affects the AML phenotype. Here we analyzed the prognostic value of RUNX1 expression levels in AML patients undergoing allogeneic hematopoietic stem cell transplantation (HCT) after non-myeloablative conditioning (NMA). Patients & Methods: We analyzed 132 patients (median age 64 years [y], range 38-75y) with diagnostic bone marrow material available who received NMA-HCT (3x30mg/m2 Fludarabine on days -4 to -1 & 2Gy total body irradiation on day 0 followed by infusion of granulocyte-colony stimulating factor mobilized peripheral blood stem cells) at our institution between 2000 and 2012. Donors were human leukocyte (HLA)-matched related (15%) or HLA-matched (61%) or mismatched (24%) unrelated. 63 (48%) patients had a normal karyotype. Presence of FLT3 -ITD or FLT3 -TKD, mutational status of IDH1, IDH2, CEBPA, NPM1 and DNMT3A as well as expression levels of mir-9, mir-181a, BAALC, ERG and MN1 were determined at diagnosis. European LeukemiaNet cytogenetic classification was: 26% favorable, 27% intermediate-I, 20% intermediate-II and 27% adverse. RUNX1 expression at diagnosis was determined by qRT-PCR and normalized to 18S as internal standard. The median cut was used to define highand low RUNX1 expressers. Median follow-up was 3.9y for patients alive. Results: High RUNX1 expressers were more likely to have de novo AML (73% vs. 52%; P =.019) and higher % blasts in peripheral blood (median 38% vs. 19%; P =.004) and bone marrow (median 68% vs. 50%; P =.002) at diagnosis. Patients with high RUNX1 expression more often had IDH1 mutations by trend (P =.061) while there was no difference in IDH2 mutation frequency (P =.32). High RUNX1 expressers showed significantly higher ERG (P <.001), MN1 (P =.005) and mir-181a (P =.002) expression. High RUNX1 expression associated with longer overall (OS, P =.065) and event free survival (EFS, P =.109) by trend in the whole cohort (Figure 1 A, B). When we restricted our analysis to patients with a normal karyotype, high RUNX1 expression associated with a significantly longer OS (P =.035) and EFS (P =.041; Figure 1 C, D), while there was no prognostic impact of RUNX1 expression in patients with an abnormal karyotype (OS, P =.606 & EFS, P =.684). In multivariate analysis high RUNX1 expression independently associated with longer OS (Hazard Ratio 0.47 [95% Confidence Interval: 0.23 - 0.96]; P =.039) and longer EFS by trend (Hazard Ratio 0.57 [95% Confidence Interval: 0.29 - 1.14]; P =.113) in patients with normal karyotype. Conclusion: Our results revealed that high expression of the hematopoietic master regulator R UNX1 at diagnosis independently associated with survival in AML patients with normal karyotype receiving NMA-HCT. High RUNX1 expression associated with distinct clinical and molecular markers. Assessing pretreatment RUNX1 levels may help to refine risk stratification in AML patients undergoing NMA-HCT. Figure 1. Figure 1. Disclosures Franke: Novartis: Other: Travel Costs; MSD: Other: Travel Costs; BMS: Honoraria. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.
Introduction: Minimal residual disease (MRD) monitoring after achievement of hematologic complete remission (CR) in patients (pts) with acute myeloid leukemia (AML) aids in identifying pts at risk of relapse. Evaluating MRD markers for clinical practice is crucial for improving outcomes in AML. Since mutations in the gene encoding Nucleophosmin (NPM1 mut) occur early in leukemogenesis & are relatively stable during disease course, they represent suitable markers for MRD monitoring. Approximately 30% of AML pts harbor NPM1 mut of which approximately 80% are type A. Some studies already demonstrated the feasibility to use NPM1 mut as MRD markers in AML pts and showed NPM1 mut MRD positivity at different time-points during disease course to be predictive for AML relapse. Hematopoietic stem cell transplantation (HCT) is a consolidating therapy option offering potential cure to AML pts. Here, we tested the feasibility to use NPM1 mut type A as MRD marker in pts receiving HCT applying digital droplet polymerase chain reaction (ddPCR) methodology. This novel technique offers high sensitivity, specificity & absolute quantification without the need of standard curves, promising inter-laboratory comparability. At the same time cost effectiveness may be superior to standard qualitative real-time PCR making ddPCR a promising new application. Methods: We analyzed 134 pts (median age 64 years [y], range 38-76y) who received HCT after non-myeloablative conditioning (NMA, 3x30mg/m2 Fludarabine on days -4 to -1 & 2Gy total body irradiation) in CR in our institution between 2000 & 2013. Donors were human leukocyte antigen (HLA)-matched related (n=23; 17.1%) or HLA-matched (n=79; 63.7%) or mismatched (≥1 antigen; n=32; 23.9%) unrelated. Median follow-up was 4.3y for pts alive. NPM1 mut status was assessed in diagnostic bone marrow (BM) samples by Sanger sequencing of the mutation hot spot in exon 12. All pts with NPM1 mut type A & available BM samples at the time of NMA-HCT (within 28 days before HCT) were analyzed by ddPCR. cDNA was applied to a duplex assay measuring NPM1 wild type (wt) & mutation type A simultaneously using fluorescent labeled probes. The mutation burden, defined as % mut type A copies/wt copies ratio, was normalized to ABL. Samples with NPM1 mut type A burden >0.01% were defined as MRD positive (MRD+) & samples with mutation burden ≤0.01% or <3 positive droplets were defined as negative according to manufacturer's recommendations. Results: We identified 42 AML pts (24.8%) with NPM1 mut of whom 24 pts had a type A mutation & BM samples at the time of NMA-HCT available. In this set 16 pts (66.7%) were classified to have favorable risk according to European LeukemiaNet (ELN) classification, 7 pts intermediate-I (29.2%) & 1 patient intermediate-II (4.1%) risk. 8 of the 16 pts harbored a FLT3 -ITD. In 4 pts NPM1 mut type A MRD was detectable at the time of NMA-HCT of which 3 pts experienced relapse. The fourth patient died 3 months after NMA-HCT due to transplantation-related complications & no further MRD time-points were available for analysis. In our cohort all clinical relapses occurred within 6 months after HCT. Two pts relapsed without detectable NPM1 mut type A at NMA-HCT. For these two pts there were no follow-up samples for further MRD monitoring after NMA-HCT available. MRD+ status at the time of NMA-HCT associated with a significantly lower cumulative incidence of relapse (P=0.001, Figure 1A) & shorter overall survival (P=0.003, Figure 1B). Conclusion: Our data demonstrate that ddPCR is a feasible method to determine the NPM1 mut type A burden by absolute quantification. Additionally, we showed the applicability of NPM1 mut type A as MRD marker in AML pts at the time of NMA-HCT. 3 out of 5 pts with clinical relapse were MRD+ at NMA-HCT. Assessing the NPM1 mut type A MRD status at the time of NMA-HCT & possibly at later time-points after HCT by ddPCR might help to identify pts at high risk of relapse. Our results strengthen the observation to use NPM1 mut for MRD monitoring. Consequently, we aim at expanding the applied ddPCR methodology to other NPM1 mut types & testing the validity in the context of clinical studies at various time points before & after NMA-HCT. Figure 1. Figure 1. Disclosures Franke: Novartis: Other: Travel Costs; MSD: Other: Travel Costs; BMS: Honoraria. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.
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