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

Cross-species microRNA/mRNA cardiac atlas.

<p>Data sets obtained for rat, dog and cynomolgus monkey are indicated. Inset: magnified view of a papillary muscle.</p

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

Negative correlation of cardiac tissue microRNA and target mRNA profiles.

<p>Timp3 and miR-1 (A), Rbm24 and miR-125b-5p (B), Tgfbr2 and miR-204 (C), Csnk2a2 and miR-208b (D). Green curve represents log2 normalized intensity for the indicated probe set. Black curve represents log2 scaled and normalized microRNA read counts. Three replicates are plotted for each structure. A, apex; LA, left atrium; LV, left ventricle; PM, papillary muscle; RA, right atrium; RV, right ventricle; S, septum; VM, valve.</p

Number of microRNAs (A) and mRNAs (B) preferentially expressed in cardiac valves or myocardium across species.

<p>Myocardium includes apex, ventricles, septum and papillary muscles. Fold change thresholds were > = 2 for microRNAs in (A), and > = 5 from mRNAs in (B).</p

Distribution of microRNAs and mRNAs in rat cardiac structures.

<p>(A) Cardiac samples are grouped according to the structure using microRNA signatures (top 10%) in a hierarchical Euclidean-linkage clustering based on miRBase17 mapping (B) Principal Component Analysis of all microRNA profiles based on miRBase17 mapping. (C) Principal Component Analysis of all mRNA profiles. Red circles: myocardial tissue (apex, left and right ventricle, septum, papillary muscle). Blue circles: left and right atrium. Grey circles: cardiac valves. A, apex. LA, left atrium. LV, left ventricle. PM, papillary muscle. RA, right atrium. RV, right ventricle. S, septum. V, valves. Individual animals are indicated by symbols.</p

Distribution of miR-1, miR-125b-5p, miR-204 and miR-208b in cardiac structures across species.

<p>Axes represent fold change vs. apex. A, apex; LA, left atrium; LV, left ventricle; PM, papillary muscle; RA, right atrium; RV, right ventricle; S, septum; V, valve.</p