Cardiac Disease Status Dictates Functional mRNA Targeting Profiles of Individual MicroRNAs

Abstract

Background: MicroRNAs are key players in cardiac stress responses, but the mRNAs, whose abundance and translational potential are primarily affected by changes in cardiac microRNAs, are not well defined. Stimulus-induced, large-scale alterations in the cardiac transcriptome, together with consideration of the law of mass action, further suggest that the mRNAs most substantively targeted by individual microRNAs will vary between unstressed and stressed conditions. To test the hypothesis that microRNA target profiles differ in health and disease, we traced the fate of empirically determined miR-133a and miR-378 targets in mouse hearts undergoing pressure overload hypertrophy.

Methods and results: Ago2 immunoprecipitation with RNA sequencing (RNA-induced silencing complex sequencing) was used for unbiased definition of microRNA-dependent and microRNA-independent alterations occurring among ≈13 000 mRNAs in response to transverse aortic constriction (TAC). Of 37 direct targets of miR-133a defined in unstressed hearts (fold change ≥25%, false discovery rate <0.02), only 4 (11%) continued to be targeted by miR-133a during TAC, whereas for miR-378 direct targets, 3 of 32 targets (9%) were maintained during TAC. Similarly, only 16% (for miR-133a) and 53% (for miR-378) of hundreds of indirectly affected mRNAs underwent comparable regulation, demonstrating that the effect of TAC on microRNA direct target selection resulted in widespread alterations of signaling function. Numerous microRNA-mediated regulatory events occurring exclusively during pressure overload revealed signaling networks that may be responsive to the endogenous decreases in miR-133a during TAC.

Conclusions: Pressure overload-mediated changes in overall cardiac RNA content alter microRNA targeting profiles, reinforcing the need to define microRNA targets in tissue-, cell-, and status-specific contexts.

Keywords: Ago2 protein; RNA; RNA-induced silencing complex; gene expression; messenger; microRNAs; regulation.

Figure 1

Effect of pressure overload on RISC-bound and global mRNAs. A) Top, representative Ago2 immunoprecipitation from mouse heart; center, global microRNA vs RISC-bound (Ago2-immunoprecipitated) microRNA abundances for 300 detected cardiac microRNAs; bottom, global mRNA vs RISC-bound (Ago2-immunoprecipitated) mRNA abundances for 11,158 detected cardiac mRNAs in nontransgenic, unstressed mice. RpM, reads per million mapped reads (microRNAs); FPKM, fragments per exon of transcript per million mapped reads (mRNAs). B) Histogram of log₂-transformed RISC ratios (abundance in RISC / abundance in global mRNA) in nontransgenic, sham-operated mice. Lower panels show log₂ ratio positions of mRNAs suppressed or derepressed in a microRNA-dependent manner after 1 week TAC (FDR<0.02).

Figure 2

Effect of pressure overload on previously described mRNA targets of regulated microRNAs. RISC-bound and global mRNA levels from 1 wk TAC hearts are shown in a standardized heatmap, for microRNA-mRNA regulatory pairs described in previous reports. Red = increased mRNA abundance in RISC-bound or global fractions, blue = decreased RNA abundance. MicroRNAs observed to be regulated at 1 wk TAC in our studies are shown in bold, and mRNAs that exhibit predicted behavior are also shown in bold. * denotes that mRNA exhibits behavior in TAC consistent with previous report(s) but proposed regulatory microRNA does not change in the same way (e.g. miR-24-3p, Jph2). # denotes that mRNA exhibits transcript abundance consistent with previous reports in TAC, but does not appear to be dependent on proposed regulatory microRNA (Grb2, Mapk1). mRNA targets of microRNAs are drawn from the following studies: miR-1a-3p^{, ,} ; miR-21-3p^, ; miR-24-3p^, ; miR-25-3p; miR-34-5p family^, ; miR-101a-3p; miR-133a-3p^{, ,} ; miR-199b-5p; miR-208a-3p^, ; miR-212/132-3p; miR-378-3p^{, ,} ; miR-499-5p.

Figure 3

Effect of sustained pressure overload on RISC-bound and global mRNAs. A) ~2300 regulated mRNAs (FDR<0.02) in either RISC-bound (upper panel) or global mRNA fractions (lower panel) at either 1 week or 2 week TAC, displayed as standardized heatmaps. Each column represents an individual heart; signals were transformed such that the mean value for sham-operated hearts is equal to 1. Row order of mRNAs in the upper panel is not the same as in the lower panel. 3 sham and 5 TAC hearts are shown for the 1 week condition; 3 sham and 7 TAC hearts are shown for the 2 week condition. B) MicroRNA-dependent, suppressed and derepressed mRNAs shown according to unstressed RISC ratio values (similarly to Figure 1b) but for 2 week TAC (FDR<0.02). C) Typical mRNA markers of cardiac hypertrophy evaluated at 1 and 2 week TAC via mRNA-sequencing (* = FDR<0.02 compared to sham); FPKM, fragments per exon of transcript per million mapped reads.

Figure 4

Cardiomyocyte and nonmyocyte distribution of TAC-regulated mRNAs. A) Pie charts of global mRNAs regulated at FDR<0.02 after 1 week TAC. Slices in each pie show cardiomyocyte (CM)-enriched (red), nonCM-enriched (green), and non-cell type-enriched mRNAs (gray). B) as for A), but for 2 week TAC.

Figure 5

Endogenous miR-133a and -378 decrease in TAC and transgene overexpression. A) RT-qPCR measurement of miR-133a and -378 during sustained TAC (normalized to U6 expression). White bars, sham; black bars, TAC. B) RT-qPCR measurement of microRNA precursors and mature microRNAs during 1 week TAC (for miR-133a) and 2 week TAC (for miR-378). White bars, nontransgenic; black bars, transgenic. * P<0.05 relative to sham (unpaired t-test); † P<0.05 relative to sham (Mann-Whitney). Numbers in bar plots designate biological replicates. Different mouse hearts were used in nontransgenic sham and TAC conditions shown in panel A to those used in panel B.

Figure 6

TAC effect on microRNA targets identified from RISC-sequencing in unstressed hearts. A–D) Venn analyses of direct (left panels) and indirect mRNA targets (right panels) of miR-133a in unstressed hearts (yellow circles), compared to mRNAs regulated in a RISC-dependent or RISC-independent manner in TAC αMHC-miR-133a hearts (red circles). E–H) As for A–D), but in αMHC-miR-378 hearts and with blue circles denoting miR-378 targets in unstressed hearts. mRNAs designated as ‘tracking’ mRNAs (not affected by TAC alone in panels A, C, E and G) are evaluated for similar regulation during TAC in the presence of the microRNA transgene (panels B, D, F and H).

Figure 7

Effect of miR-378 overexpression on cardiac contractile performance and TAC response. A) Myocyte cross-sectional area (from ~500 cells/heart) and length (from ~100 cells/heart); representative images from nontransgenic hearts are shown. B) M-mode echocardiography in awake mice (n=6–10 per group) and dobutamine-stimulated contractility in anesthetized mice, n=4 mice per group; P<0.0001 between genotypes by 2-way ANOVA with Sidak multiple correction test. C) β-adrenergic pathway and contractile function mRNAs; heatmap colors show log₂fold changes relative to nontransgenic mean. Significantly regulated mRNAs (fold-change 25%, FDR<0.1) are in bold. D) Representative M-mode echocardiograms and E) echocardiographic parameters determined from TAC studies. F) Myocyte cross-sectional area and lengths as for A). White bars, nontransgenic; black bars, αMHC-miR-378. * = P<0.05, † = P<0.1, unpaired 2-tailed t-test; ‡ = P<0.05 relative to previous time point; n=7–8 per group.