Perturbation of microRNAs in Rat Heart during Chronic Doxorubicin Treatment

<div><p>Anti-cancer therapy based on anthracyclines (DNA intercalating Topoisomerase II inhibitors) is limited by adverse effects of these compounds on the cardiovascular system, ultimately causing heart failure. Despite extensive investigations into the effects of doxorubicin on the cardiovascular system, the molecular mechanisms of toxicity remain largely unknown. MicroRNAs are endogenously transcribed non-coding 22 nucleotide long RNAs that regulate gene expression by decreasing mRNA stability and translation and play key roles in cardiac physiology and pathologies. Increasing doses of doxorubicin, but not etoposide (a Topoisomerase II inhibitor devoid of cardiovascular toxicity), specifically induced the up-regulation of miR-208b, miR-216b, miR-215, miR-34c and miR-367 in rat hearts. Furthermore, the lowest dosing regime (1 mg/kg/week for 2 weeks) led to a detectable increase of miR-216b in the absence of histopathological findings or alteration of classical cardiac stress biomarkers. <em>In silico</em> microRNA target predictions suggested that a number of doxorubicin-responsive microRNAs may regulate mRNAs involved in cardiac tissue remodeling. In particular miR-34c was able to mediate the DOX-induced changes of Sipa1 mRNA (a mitogen-induced Rap/Ran GTPase activating protein) at the post-transcriptional level and in a seed sequence dependent manner. Our results show that integrated heart tissue microRNA and mRNA profiling can provide valuable early genomic biomarkers of drug-induced cardiac injury as well as novel mechanistic insight into the underlying molecular pathways.</p> </div

Chronic DOX treatment (3 mg/kg/week) alters levels of 25 microRNAs from week 2 onwards.

<p>Variation of cardiac microRNA levels versus vehicle are reported for animals treated with DOX 3 mg/kg/week for 2 and 4 weeks. Values were calculated via the relative quantification (ΔΔCt) method by using the mammalian U6 snRNA as a normalizer. MicroRNAs showing same trend at 2 and 4 weeks are italicized. # indicates microRNAs selected for further analysis. Significant P values (<0.05) are in bold. FC  =  fold change.</p

Study design and representative micrograph showing DOX-related vacuolation in the myocardium.

<p>(A) Six adult male rats were injected with the indicated doses of vehicle, doxorubicin (DOX), dexrazoxane (DZR), etoposide (EPS) or a combination of DOX and DZR for 2, 4 or 6 weeks. Cardiac tissue was excised and deep frozen for gene expression and microRNA profiling experiments. A representative micrograph of a toluidine blue stained myocardial section of a control (B) and of a DOX treated animal (C). Black arrows indicate sarcoplasmic micro- and macro- vacuolation of cardiomyocytes.</p

DOX 3 mg/kg/week altered levels of genomic cardiomyopathy indicators (Ankrd/Carp, Nppb, Myh7 and Myh6).

<p>Expression fold change relative to vehicle were represented for DOX 3 mg/kg/week at 2, 4 and 6 weeks time point (n = 6) for (A) Ankrd/Carp, (B) Nppb, (C) Myh7 and (D) Myh6. For each time point and each probe set, vehicle values were averaged and normalized to 1. The same correction was applied to the DOX treated values. Affymetrix probe-set number is indicated in brackets. Error bars represent standard deviation. T-test was performed for vehicle- vs. DOX-treated at each time point. *P<0.05, **P<0.01, ***P<0.005, NS = Non-Significant. (No t-test for #, as n = 2).</p

Ambra1 expression was induced by DOX treatment <i>in vivo</i> and miR-34c could control its endogenous expression levels in H9c2 cardiac myoblasts.

<p>(A) Fold change of Ambra1 probe set in rat heart tissue treated with DOX. Fold change and statistical significance were assessed <i>vs.</i> each vehicle group. n = 4 to 6 (except #, n = 2) (B) Endogenous levels of Ambra1 were measured after miR-34c over-expression (miR-34c mimic) or inhibition (miR-34c HI) in absence of presence of DOX 0.1. Fold change value were normalized <i>vs.</i> the respective negative transfection controls in the untreated condition (n = 3). *P<0.05, **P<0.01, ***P<0.005. FC  =  fold change.</p

miR-34c directly controls DOX-induced Sipa1 mRNA decrease.

<p>(A) Sipa1 mRNA raw expression values decreased in the heart of rats treated with DOX. #, n = 2. (B) DOX treatment for 24 h caused a decrease of Spa1 mRNA and an increase of miR-34c in cardiac myoblast cells (H9c2). (C) H9c2 endogenous Sipa1 mRNA was decreased by transfection of miR-34c mimic, and increased using a miR-34c hairpin inhibitor (HI). Transfection with miR-34c mimic and inhibitor respectively exacerbated and rescued Sipa1 mRNA levels in H9c2 treated with DOX 0.1 and 1 µM overnight in comparison to negative controls. (D) Alignment of mammalian miR-34 family. Capital letters indicate mismatch in the sequence. Sipa1 3′-UTR wt and MUT construct: 12 nt surrounding the predicted seed are shown. HEK 293 cells were co-transfected with pmiR-GLO-Sipa1 and the indicated miRNA mimic or a <i>C. elegans</i> negative control mimic. Averaged and normalized Renilla luciferase signal was obtained from 3 independent experiments, each run in quadruplicate. Y axis represents percentual residual luciferase activity in the indicated conditions. Mutant 3′ UTR restores luciferase activity in Sipa1/miR-34c. *P<0.05, **P<0.01, ***P<0.005.</p

Relative quantification of DOX-responder microRNAs in rat heart across all groups.

<p>Relative quantification of (A) miR-208b, (B) miR-215, (C) miR-216b, (D) miR-367 and (E) miR-34c in DOX, DOX + DZR, EPS groups, normalized versus vehicle treated animals. Expression levels were measured by single assay qPCR (n = 3, except #, n = 2). DOX: Doxorubicin, DZR: dexrazoxane, EPS: etoposide; numbers indicate the weekly dose of each compound in mg/kg/week. Empty spaces represent non-sampled animals. The vehicle treated is the first column of each time-point. The animals used in this experiment were distinct from the ones represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040395#pone-0040395-t001" target="_blank">Table 1</a>. Error bars represent SD. T-test results are indicated by asterisks for significant DOX-treated groups vs. their own vehicle-treated, unless otherwise specified by horizontal range bars; *P<0.05, **P<0.01, ***P<0.005, NS = Non-Significant).</p

DOX-induced upregulation of miR-216 and miR-367 expression precedes the detection of overt histopathological lesions in cardiac tissue.

<p>Blue bars show cumulative vacuolation grade. X axis shows the DOX regimen in mg/kg/week received by the animals at 2 weeks. Y axes report cumulative histopathological scores and microRNA fold change vs. untreated cardiac tissues (normalized at value 1). Path grading  =  cumulative vacuolation score. FC  =  fold change. SEM  =  standard error on the mean.</p

Heart Structure-Specific Transcriptomic Atlas Reveals Conserved microRNA-mRNA Interactions

<div><p>MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level and play key roles in heart development and cardiovascular diseases. Here, we have characterized the expression and distribution of microRNAs across eight cardiac structures (left and right ventricles, apex, papillary muscle, septum, left and right atrium and valves) in rat, Beagle dog and cynomolgus monkey using microRNA sequencing. Conserved microRNA signatures enriched in specific heart structures across these species were identified for cardiac valve (miR-let-7c, miR-125b, miR-127, miR-199a-3p, miR-204, miR-320, miR-99b, miR-328 and miR-744) and myocardium (miR-1, miR-133b, miR-133a, miR-208b, miR-30e, miR-499-5p, miR-30e*). The relative abundance of myocardium-enriched (miR-1) and valve-enriched (miR-125b-5p and miR-204) microRNAs was confirmed using in situ hybridization. MicroRNA-mRNA interactions potentially relevant for cardiac functions were explored using anti-correlation expression analysis and microRNA target prediction algorithms. Interactions between miR-1/Timp3, miR-125b/Rbm24, miR-204/Tgfbr2 and miR-208b/Csnk2a2 were identified and experimentally investigated in human pulmonary smooth muscle cells and luciferase reporter assays. In conclusion, we have generated a high-resolution heart structure-specific mRNA/microRNA expression atlas for three mammalian species that provides a novel resource for investigating novel microRNA regulatory circuits involved in cardiac molecular physiopathology.</p> </div

Anti-correlated microRNA targets are directly inhibited by microRNA over expression.

<p>(A–D) Real-Time RT-PCR of Timp3, Rbm24, Tgfbr2 and Csnk2a2 in HPASM cells transfected with mimics for miR-1, miR-125b-5p, miR-204, miR-499 and miR-208b or with a mimic microRNA negative control. Data were normalized to 18S RNA. (E-H) Luciferase activity of wild-type (WT) or mutant (MUT) Timp3, Rbm24, Tgfbr2 and Csnk2a2 3′-UTR reporter genes cotransfected with their paired microRNA mimics. Data were averaged for n = 4 in 3 independent experiments and error bars represented standard deviation of the mean. P values: * = P<0.05, ** = P<0.01, *** = P<0.005.</p