Direct and indirect involvement of microRNA-499 in clinical and experimental cardiomyopathy

Abstract

Rationale: MicroRNA-499 and other members of the myomiR family regulate myosin isoforms in pressure-overload hypertrophy. miR-499 expression varies in human disease, but results of mouse cardiac miR-499 overexpression are inconsistent, either protecting against ischemic damage or aggravating cardiomyopathy after pressure overload. Likewise, there is disagreement over direct and indirect cardiac mRNAs targeted in vivo by miR-499.

Objective: To define the associations between regulated miR-499 level in clinical and experimental heart disease and modulation of its predicted mRNA targets and to determine the consequences of increased cardiac miR-499 on direct mRNA targeting, indirect mRNA modulation, and on myocardial protein content and posttranslational modification.

Methods and results: miR-499 levels were increased in failing and hypertrophied human hearts and associated with decreased levels of predicted target mRNAs. Likewise, miR-499 is increased in Gq-mediated murine cardiomyopathy. Forced cardiomyocyte expression of miR-499 at levels comparable to human cardiomyopathy induced progressive murine heart failure and exacerbated cardiac remodeling after pressure overloading. Genome-wide RNA-induced silencing complex and RNA sequencing identified 67 direct, and numerous indirect, cardiac mRNA targets, including Akt and MAPKs. Myocardial proteomics identified alterations in protein phosphorylation linked to the miR-499 cardiomyopathy phenotype, including of heat shock protein 90 and protein serine/threonine phosphatase 1-α.

Conclusions: miR-499 is increased in human and murine cardiac hypertrophy and cardiomyopathy, is sufficient to cause murine heart failure, and accelerates maladaptation to pressure overloading. The deleterious effects of miR-499 reflect the cumulative consequences of direct and indirect mRNA regulation, modulation of cardiac kinase and phosphatase pathways, and higher-order effects on posttranslational modification of myocardial proteins.

Figure 1. miR-499 upregulation and target suppression in human heart failure and murine genetic cardiomyopathy

(A) Increased miR-208, miR-499 levels in human heart failure; white = non-failing (Ctrl), blue = failing hearts (HF), red = hypertrophied non-failing hearts (Hyp). The miR expression arrays used did not distinguish between miRs-208a and -208b. Individual data are shown together with mean ± s.d. (B) Increased Myh7b levels in human heart failure; white = non-failing (Ctrl) and blue = failing hearts (HF). (C) Downregulation of 98 miR-499 target mRNAs, predicted by TargetScan, in human heart failure (greater than 1.2-fold downregulation, P<0.05, Online Table I). Four representative hearts each are shown for non-failing (NF), and failing hearts with cardiomyopathy of ischemic origin (isch) or non-ischemic origin (non-isch). Red = higher expression, blue = lower expression. (D) miR microarray analysis of Gαq transgenic hearts , displaying miRs according to degree of upregulation (1.2-fold or greater, P<0.05; miR-451, 4.4 fold-upregulated in Gq, is at the top). (E) miR-499 analyzed by RT-qPCR relative to combined reference of 5S rRNA, U6 snRNA and Gapdh mRNA in nontransgenic (n=3) and Gαq hearts (n=6). Data shown are mean +/minus; s.e.m., arbitrary units (nontransgenic = 1); * denotes P=0.0006, 2-tailed unpaired t-test. (F) Downregulation of 13 miR-499 target mRNAs, predicted by TargetScan (greater than 1.2-fold downregulation, P<0.05) in Gαq transcriptomes analyzed by RNA-sequencing . Red, higher expression; blue, lower expression. * denotes downregulation of the same mRNA in human heart failure.

Figure 2. Cardiac miR-499 overexpression causes heart failure in mice

(A) Cardiac-directed overexpression of miR-499 in two transgenic mouse lines at levels similar to human heart failure (TG-16 and TG-15) and in a higher-overexpressing line (TG-53). * denotes P<0.0001 relative to nontransgenic (1-way ANOVA); # denotes significant difference to all other groups (Tukey’s pairwise comparisons). (B) Progressive dilated cardiomyopathy in miR-499 transgenic hearts shown by increases in left-ventricular end-diastolic dimension (LVEDD) and decreases in fractional shortening (FS%) at 20 weeks of age. Two-way ANOVA was used to assess effects of timepoint and genotype (full details in Online Table II); * denotes significant difference vs age-matched nontransgenic. For both (A) and (B), data shown are mean ± s.e.m.; numbers of hearts are shown in columns. (C) Representative M-mode echocardiograms from 20 week-old mouse hearts. (D) Myocyte cross-sectional area shown by fluorescent wheat germ agglutinin staining of 20 week-old mouse hearts. White scale bar represents 10 μm. Quantitation represents mean values from ~600 myocytes, from each of two hearts. * denotes P=0.0004 relative to nontransgenic (1-way ANOVA).

Figure 3. Transcriptome regulation in miR-499 transgenic mice

(A) Markers of the hypertrophic / fetal recapitulation gene program; α-cardiac actin (Actc1), SERCA2a (Atp2a2), α-MHC (Myh6), β-MHC (Myh7), α-skeletal actin (Acta1) and ANP (Nppa) in 4 week-old hearts. (B) Heatmap of 136 mRNAs indirectly regulated by miR-499 in 4 week-old and 8 week-old transgenic hearts (details in Online Table III). (C) Downregulation of 31 miR-499 target mRNAs predicted by TargetScan in miR-499 transgenic hearts (greater than 1.2-fold downregulation, P<0.05, Online Table IV). * denotes downregulation of the same mRNA in human heart failure; + denotes downregulation of the same mRNA in Gαq mice (Figure 1). Underlined mRNAs are those confirmed by RT-qPCR in panel D. Red = higher expression, blue = lower expression. (D) TaqMan RT-qPCR validation of 3 predicted miR-499 target mRNAs. Expression level is normalized to a combined reference of Actb, Gapdh and Hmbs. Relative abundances of Irs2, Rcn2 and Tmbim6 correlate to RNA-sequencing determinations (Online Table IV). * denotes P<0.05, unpaired 2-tailed t-test.

Figure 4. miR-499 overexpression worsens the response to pressure overload in mice

(A) Representative sections of nontransgenic sham, nontransgenic pressure-overloaded (TAC) and miR-499 TG-16 pressure-overloaded hearts 6 weeks after surgery. (B) Development of hypertrophy shown by echocardiography of nontransgenic (n=5) and miR-499 TG-16 (n=3) animals. Data shown are mean ± s.e.m. ; * denotes significant difference at 6 weeks (P=0.03 for LVEDD and P=0.007 for FS%, 2-tailed unpaired t-test). (C) Heart weight / body weight ratios after 6 weeks TAC. * denotes significant difference (P=0.036, 1-tailed unpaired t-test). (D) Myocyte cross-sectional area shown by fluorescent wheat germ agglutinin staining. White scale bar represents 10 μm. Quantitation represents mean values from ~600 myocytes, from each of 2 hearts. * denotes P=0.04. Dotted lines, C and D: typical levels in age-matched, untreated nontransgenic mice. (E) Fibrosis after 6 weeks of TAC shown by Masson’s trichrome staining. White scale bar represents 10 μm. (F, left) Principal components analysis of 6,500 mRNAs (those expressed at >=3 copies/cell) in nontransgenic and miR-499 TG-16 hearts, sham-treated and after 1 week of TAC (5 hearts per treatment). Individual hearts are shown as filled spheres; wireframes enclose all hearts of a treatment group. Reduction of variance to 3 principal components (the 3 dimensions shown in the plot) accounts for 49.8% of the total variance in the data set.

Figure 5. Direct targets of miR-499 in mouse hearts

(A, left) Fold-change in RISC score (RISCome expression/transcriptome expression) between transgenic and nontransgenic mice for 63 direct targets of miR-499 identified by RISC-sequencing. (A, right) Change in RISCome expression for a further 4 miR-499 targets. (B) Fold-change in transcriptome expression for the miR-499 targets shown in (A). nc = no change. Black bars, miR-499 transgenic TG-16; gray bars, miR-499 transgenic TG-53. (C) Schematic depiction of miR-499 recognition sites in miR-499 targets defined by RISC-sequencing. Graph on the left shows number of sites according to mRNA position; graph on the right shows aggregate counts. The majority (68%) of putative binding sites are in the 3′ untranslated region (UTR), with 31% in coding regions and 1% in the 5′ UTR.

Figure 6. Similar and differential regulation of mRNA and protein for indirect targets of miR-499

Upper panel: Representative 2D DiGE gel image. Green = miR-499, red = nontransgenic cardiac protein. Lower panel: Comparison of mRNA and protein regulation for 22 gene products. Mybpc3 (MyBP-C), Ckmt2, Mdh1, P4hb and Pdha1 migrated as multiple species and were not analyzed. Also identified, but with neither mRNA nor protein regulation: Acadvl, Anxa6, Cox5a, Hspa8, Hspb8, Myl7, Vdac2, Vim. White bars, mRNA regulation; black bars, protein regulation. nc = no change. Regulated mRNAs had changes of ≥20%, P<0.05. Proteins were classed as regulated if changes of ≥20% in the same direction were identified on at least 3 of 5 gels.

Figure 7. Differential phosphorylation in nontransgenic and miR-499 transgenic mice at 1 week of pressure overload

(A) Representative phosphoproteome 2D gel image of nontransgenic sham-treated cardiac protein samples. Green is total protein stain; red is ProQ-diamond phosphoprotein; rectangle, HSP90β (H); circle, PP1α (P). (B) 2D immunoblots of HSP90β and PP1α; 1D SDS-PAGE of a nontransgenic, sham-treated cardiac protein sample is shown to at right. White squares denote regions shown in panels C and D. (C) Left: magnified view of 2D immunoblot for HSP90β. Right: same region in representative phosphoproteome 2D gels for nontransgenic and TG-16, sham- and TAC-treated hearts. (D) as for (C) but for PP1α. A total of 5 hearts were used in each treatment group (Online Figure VI).

Figure 8. Signaling networks influenced by miR-499

MetaCore was used to explore signaling pathways involving direct and indirect targets of miR-499. Dotted lines, indirect action; solid lines, direct action (arrowheads denote activation, while bars denote inhibition). miR-499 targets are in bold, and those with altered phosphorylation (HSP90β and PP1α) are in blue.