microRNA-22 promotes heart failure through coordinate suppression of PPAR/ERR-nuclear hormone receptor transcription

Abstract

Increasing evidence suggests that microRNAs are intimately involved in the pathophysiology of heart failure. MicroRNA-22 (miR-22) is a muscle-enriched miRNA required for optimum cardiac gene transcription and adaptation to hemodynamic stress by pressure overload in mice. Recent evidence also suggests that miR-22 induces hypertrophic growth and it is oftentimes upregulated in end stage heart failure. However the scope of mRNA targets and networks of miR-22 in the heart failure remained unclear. We analyzed transgenic mice with enhanced levels of miR-22 expression in adult cardiomyocytes to identify important pathophysiologic targets of miR-22. Our data shows that forced expression of miR-22 induces a pro-hypertrophic gene expression program, and it elicits contractile dysfunction leading to cardiac dilation and heart failure. Increased expression of miR-22 impairs the Ca(2+) transient, Ca(2+) loading into the sarcoplasmic reticulum plus it interferes with transcription of estrogen related receptor (ERR) and PPAR downstream genes. Mechanistically, miR-22 postranscriptionally inhibits peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), PPARα and sirtuin 1 (SIRT1) expression via a synergistic circuit, which may account for deleterious actions of unchecked miR-22 expression on the heart.

Figure 1. Enforced expression of miR-22 in the heart is sufficient to induce cardiomyopathy.

(A) Representative hearts of TG-M, TG-H and non-transgenic (WT) mice at 12-weeks of age. Top and bottom panels show Masson’s Trichrome stained sections at low and high magnification respectively (Scale bars: 1mm and 50mm). (B,C) Heart weight normalized to body weight (HW/BW) in 12-week old TG-M and TG-H mice versus WT (n = 5-12). (D) Analyses of cardiac function by echocardiography in 12-week old TG-M and WT mice. LVESD, LV end-systolic dimension; LVEDD, LV end-diastolic dimension; LVPWS, LV posterior wall thickness at end-systole; FS, fractional shortening (n = 10-11). (E) Hemodynamic analysis with a Millar catheter in 12-week old TG-M and WT mice. HR, heart rate; LVEDP, LV end-diastolic pressure; LVP, maximal LV pressure; +dP/dt max, maximal contraction rate; -dP/dt max, maximal relaxation rate (n = 5). (F) Relative mRNA expression levels of indicated genes in 12-week old TG-M and WT mice. Bars represent expression normalized with WT set equal to 1.0 (n = 3-4). Student t test. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 2. miR-22 co-represses PGC-1α, PPARα, and SIRT1 in the heart.

(A) Pgc1a, Ppara and Sirt1 mRNA was quantified by real-time PCR from ventricles of 5- (5w) or 12-week (12w) old mice of indicated genotypes. Bars represent expression normalized with WT set equal to 1.0 (n = 3-4). (B) Representative Western blot and quantitation of PGC-1α, PPARα, and SIRT1 levels from ventricular lysates obtained from 12-week old TG-H and WT mice. GAPDH was used as a loading control (n = 3 immunoblots from three mice each genotype). (C) Pgc1a, Ppara and Sirt1 each contain highly conserved miR-22-mRNA interaction motifs within their 3’ UTRs. (D) Luciferase activity in 3T3 MEF cells transfected with indicated wt or site mutant (mut) 3’ UTR reporter constructs in the presence of miR-22 or control miR mimic. Data are from two experiments carried out in triplicate. (E) Cardiac Pgc1a, Ppara and Sirt1 mRNA expression levels were evaluated by qPCR from 6-week old miR-22-deficient (KO) and WT mice (n = 3). Student t test, (A,B,E); or 2-way ANOVA with the Tukey post hoc test, (D). *, P<0.05; **, P<0.01;.***, P<0.001.

Figure 3. Enforced miR-22 expression impairs ERR/PPAR-dependent transcription in the heart.

(A, E, F, G, and H) Cardiac mRNA expression levels for indicated genes were quantified by qPCR in TG-H, TG-M and WT mice aged (A) 5- or (E-G) 12-weeks old. In panels (E-H) bars represent expression normalized with WT set equal to 1.0 (n = 3-4). (B) Gene Set Enrichment Analysis (GSEA) of sorted gene lists obtained from the microarray detected significant expression shifts in the indicated KEGG pathways in TG-H hearts. **, P<0.005. (C and D) Transcriptome microarray-obtained expression profile heat maps for (C) ERR-regulated OXPHOS or (D) PPAR-regulated lipid metabolism genes in 12-week old TG-H and WT mice. Student t test, (E,F,G,H); or 1-way ANOVA with the Dunnett post hoc test, (A). *, P<0.05; **, P<0.01; ***, P<0.001; †, P=0.06; ‡, P=0.07.

Figure 4. Diminished sarcoplasmic reticulum (SR) Ca²⁺ load and Ca²⁺ transient in miR-22 transgenic mice.

(A) Representative traces of Ca²⁺ transients from TG-M and WT adult ventricular cardiomyocytes upon 1Hz pacing in 1.8mmol/L, Ca²⁺ Tyrode solution, and (B) after rapid exposure to 10mM caffeine. Note that the sweep velocity was constant and the time scale was identical to data in first panel. (C) Quantification of electrically evoked Ca²⁺ transients, and (E) estimation of SERCA2 activity from time constant of Ca²⁺ transient decline (τ of CaT) in TG-M and WT myocytes. (D) SR Ca²⁺ load response, and (F) NCX activity estimation during caffeine stimulation (τ of caffeine decline) in 0 Na⁺, 0 Ca²⁺ Tyrode solution (n = 3-4 mice). Student t test. *, P<0.05.