Deficiency for MicroRNA-582 does not impact dilated cardiomyopathy or heart failure induced by pressure overload in vivo

Figures

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  FIGURE 1. Expression of miR-582-5p and basic characterization of 582-KO mice. Relative expression of miR-582-5p in different in vivo ((A), mouse) and in vitro ((B), NRVCM) models of cardiomyopathy, n = 5-16 (mice), n = 2-4 experiments (NRVCM), (C) relative expression of miR-582-5p in heterozygous and homozygous 582-KO mice, (D) RNA-expression of Pde4d in male (blue) and female (red) mice, (E) Hematoxylin-eosin staining (top) and Sirius Red/Fast Green Collagen Staining (bottom), (F–I) morphometric and echocardiographic data and expression of Nppa and Nppb in male mice, (J–M) morphometric and echocardiographic data and expression of Nppa and Nppb in female mice, n = 5-9 animals, (BW, body weight; Ctrl, control; ET, endothelin 1; F, female; FS, fractional shortening; het, heterozygous; hom, homozygous; HW, heart weight; KO, knockout; M, male; PE, phenylephrine; WT, wildtype, *p < 0.05, **p < 0.01, ***p < 0.005, ns, not significant).

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  FIGURE 2. TAC surgery in male 582-KO mice (A–H) and crossbreeding CS1-KO/582-KO mice (I-R). (A) relative expression of miR-582-5p, (B–D) echocardiographic data, (E) Heart weight/body weight ratio, (F–H) expression of Nppa, Nppb and Rcan1.4 (n = 5-10 animals), (I–R) morphometric and echocardiographic data and expression of Nppa and Nppb of male (I–M) and female (N–R) CS1-KO/582-KO cross bred mice, n = 6-15 animals (BW, body weight; CS1, Calsarcin-1; HW, heart weight; KO, knockout; LVAW; d, left ventricular anterior wall thickness at end-diastole; LVID; d, left ventricular internal diameter at end diastole; TAC, transverse aortic constriction; WT, wildtype, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns, not significant).

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  FIGURE 3. Basic characterization of TG-582 mice (A–J), TAC surgery in male TG-582 mice (K–R) and crossbreeding of male CS1-KO/TG-582 mice (S-W). (A) relative expression of miR-582-5p, (B) Hematoxylin-eosin staining (top) and Sirius Red/Fast Green Collagen Staining (bottom), (C–F) morphometric and echocardiographic data and expression of Nppa and Nppb in male mice, (G–J) morphometric and echocardiographic data and expression of Nppa and Nppb in female mice, n = 3-14 animals; (K) relative expression of miR-582-5p, (L–N) echocardiographic data, (O) Heart weight/body weight ratio, (P–R) expression of Nppa, Nppb and Rcan1.4, n = 6-12 animals; (S–W) morphometric and echocardiographic data and expression of hypertrophic marker genes in male CS1-KO/TG-582 cross bred mice, n = 2-14 animals, (BW, body weight; CS1, Calsarcin-1; FS, fractional shortening; HW, heart weight; IVS; d, interventricular septal thickness at end-diastole; KO, knockout; LVID; d, left ventricular internal diameter at end diastole; LVPW; d, diastolic left ventricular posterior wall thickness at end-diastole; TAC, transverse aortic constriction; WT, wildtype, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns, not significant).

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  FIGURE 4. In silico target prediction (A) and relative RNA-expression of published (B,C) and predicted (D–J) target genes of miR-582-5p. (A) In silico target prediction employing the algorithms: MicrocCosm (miRanda), TargetScan, MiRDB (MirTarget) and DIANA-MicroT yielding 18 overlapping predicted target genes, (B–J) relative RNA-expression of previously identified miR-582-5p target genes Creb1 (B) and F2rl2 (C) and relative RNA-expression of seven predicted miR-582-5p target genes (D–J) in 582-KO and TG-582 mice, 582-KO and TG-582 mice after TAC-surgery and in CS1-KO mice crossbred with 582-KO and TG-582 mice respectively, n = 3-13. (CS1, Calsarcin-1; KO, knockout; TAC, transverse aortic constriction; WT, wildtype; ns, not significant).