Cardiomyocyte-Derived Exosomal MicroRNA-92A Mediates Post-Ischemic Myofibroblast Activation Both in Vitro and Ex Vivo

Figure 1. Functional role of miR‐92a in fibroblast activation. (A) miR‐92a targets SMAD7; complementary nucleotides between the target region of Smad7 3′ untranslated region (UTR) (in yellow) and miR‐92a are conserved across different species; Luciferase activity (right panel) was measured 48 h after transfection, and values are shown as fold change of the luciferase activity detected using the firefly vector without Smad7 3′ UTR (checkered bar); a mutated Smad7 3′ UTR (Smad7 MUT) and a cel‐miR‐39 mimic have been used as further controls. (B–E) Upregulation of miR‐92a following cardiac ischemic injury. We found a significant upregulation of miR‐92a in cardiac fibroblasts (B) and cardiomyocytes (C) and in exosomes obtained from cardiomyocytes (D,E) isolated 7 days after MI (n ≥ 6 mice/group); Smad7 mRNA was markedly downregulated while αSMA was upregulated in fibroblasts post‐MI (B). Representative immunoblots (D) showing the presence of marker proteins typically enriched in exosomes, namely, CD81, Tumor Susceptibility Gene 101 (TSG101), and syntenin‐1, and the absence of contamination from other cellular components using Heat Shock Protein 90 Beta Family Member 1 (HSP90B1, a.k.a. GRP94)…

Figure 2. Effects of cardiomyocyte‐derived exosomes on fibroblast activation. (A) Fibroblasts were incubated with exosomes obtained from cardiomyocytes isolated from SHAM and myocardial infarction (MI) mice (n ≥ 6 mice/group) 7 days post‐surgery; such incubation induced an upregulation of miR‐92a, αSMA, collagens I and III, and periostin. Mean ± SEM of at least three independent experiments (all significantly different compared with SHAM, P < 0.05). (B) Effects of conditioned cardiomyocyte medium on the activation of fibroblasts; conditioned medium from cardiomyocytes isolated (n ≥ 6 mice/group) 7 days post‐MI induced an upregulation of miR‐92a, αSMA, and periostin. However, the addition of an inhibitor of exosome release (GW4869 10 μM for 12 h) significantly attenuated these responses. Mean ± SEM of at least three independent experiments; *P < 0.05 vs. SHAM. (C,D) Effects of miR mimics and inhibitors on fibroblast activation. Conditioned medium from cardiomyocytes isolated (n ≥ 6 mice/group) 7 days post‐MI or SHAM surgery was added to fibroblasts transfected with miR‐92a mimics (C) and inhibitors (D), or negative control miR (NC). Mean ± SEM of at least three independent experiments; *P <…