Cardiosomal microRNAs Are Essential in Post-Infarction Myofibroblast Phenoconversion

Figures

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  Figure 1. Identification of miR-195 as negative regulator of SMAD7. Luciferase activity was measured 48 h after transfection, and the values are shown as fold change of the luciferase activity with respect to the empty plasmid; cel-miR-39 mimic was used a negative control (NC). Mean ± S.E.M. of at least three independent experiments; * p < 0.05 vs. NC.

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  Figure 2. Upregulation of miR-195 post MI. This miRNA was upregulated in fibroblasts (A) as well as in exosomes obtained from cardiomyocytes (cardiosomes, B) isolated seven days after MI (n ≥ 6 mice/group); Smad7 mRNA was significantly down-regulated whereas αSMA and periostin, known markers of myofibroblast activation, were upregulated in fibroblasts post-MI (A). In panel C, cardiosome preparations were re-suspended in 300 mL PBS and spiked with 20 ρmol of a synthetic oligonucleotide corresponding to the mature sequence of miR-126 (exogenous miRNA used as control); samples were then treated or not with Triton X-100 (1%) and incubated with or without RNase A (0.5 U) and T1 (15 U) for 30 min at 37 °C before RNA extraction. Mean ± S.E.M. of at least three independent experiments; * p < 0.05 vs. SHAM (panels A, B) or untreated (panel C).

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  Figure 3. Effects of cardiosomes on fibroblast activation. Fibroblasts were incubated with cardiosomes obtained from cardiomyocytes isolated from SHAM and MI mice (n ≥ 6 mice/group) seven days post-surgery; such incubation induced an upregulation of miR-195 (A), αSMA (B), collagen I and III (C) fibroblast activation protein (FAP, D), periostin (E), fibronectin containing extra domain A (Fibronectin ED-A, F), chemokine (C-X-C motif) ligand 1 (CXCL1, G), interleukin-6 (IL-6, H). Mean ± S.E.M. of at least three independent experiments; * p < 0.05 vs. SHAM.

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  Figure 4. Effects of conditioned cardiomyocyte medium on the activation of fibroblasts. Conditioned medium from cardiomyocytes isolated (n ≥ 6 mice/group) seven days post-MI induced an upregulation of miR-195, αSMA, and periostin. However, the addition of an inhibitor of cardiosome release (GW4869 10 μM for 12 h) significantly attenuated these responses. Mean ± S.E.M. of at least three independent experiments; * p < 0.05 vs. SHAM.

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  Figure 5. Effects of miR mimics and inhibitors on fibroblast activation. Conditioned medium from cardiomyocytes isolated (n ≥ 6 mice/group) seven days post-MI or SHAM surgery was added to fibroblasts transfected with miR-195 mimic (A), miR-195 inhibitor (B), or negative control miR (NC). Mean ± S.E.M. of at least three independent experiments; * p < 0.05 vs. SHAM medium + NC (Panel A) or vs. MI medium + NC (Panel B), # p < 0.05 vs. SHAM.