Cardiac fibroblasts secrete exosome microRNA to suppress cardiomyocyte pyroptosis in myocardial ischemia/reperfusion injury

Fig. 1. Pyroptosis of cardiomyocytes is involved in H/R injury. A Cell viability in H/R cardiomyocytes was measured by CCK8 kit, the time points indicate the reoxygenation time course after 6 h hypoxia. B RT-qPCR assayed ELAVL1, NLRP3, caspase-1 mRNA expression levels in H/R cardiomyocytes with 6 h hypoxia/1h reoxygenation. C and D Western blot detected the protein levels of ELAVL1, NLRP3, and cleaved caspase-1 in H/R cardiomyocytes. E and F The IL-1β and IL-18 expression levels were determined by ELISA kit. LPS was used as a positive control. n = 3, data are shown as mean ± SD. *p < 0.05, **p < 0.01 vs. the control group

Fig. 2. CFs avert pyroptosis of cardiomyocytes. H/R cardiomyocytes co-culture with CFs, GW4869 (10 uM) was used to inhibit the exosome secretion from CFs. A CCK8 kit was used to measure the cell viability. B and C Protein levels of ELAVL1, NLRP3, and cleaved caspase-1 were detected by western blot and quantitative analysis of the protein expression. D and E IL-1β and IL-18 expression was determined by ELISA kit. n = 3, data are shown as mean ± SD. *p < 0.05, **p < 0.01 vs. the control group

Fig. 3. Characterization of CF-derived exosomes. A Western blot detected the exosome protein biomarkers CD9, CD81, TSG101. B and C Exosome morphological characteristics and size were determined by transmission electron microscope and dynamic light scattering. D Fluorescence microscope was used to trace the CFs exosome location in H/R cardiomyocytes

Fig. 4. CFs reduce pyroptosis of cardiomyocytes through exosomes. A Cell viability was measured by CCK8 kit when exosome was co-cultured with H/R cardiomyocytes. B and C Western blot analysis the expression of ELAVL1, NLRP3, and cleaved caspase-1 in H/R cardiomyocytes that co-cultured with exosome. D and E ELISA assay detected the IL-1β, IL-18 protein expression in H/R cardiomyocytes that co-cultured with exosome. n = 3, data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the control group

Fig. 5. Exosome miR-133a targets ELAVL1 and reduce pyroptosis in cardiomyocytes. A Prediction miRNAs target ELAVL1 by TargetScan, miRWalk, and miRDB database. B qPCR detected miRNAs expression predicted target binding through three databases in control exosome and CFs exosome. Control exosomes secreted by 293T cells. C The putative miR-133a binding sites in 3′UTR of ELAVL1. Replacement of Adenine bases with Uracil (A to U) can be used for the construction of mutant reporter. D The relative luciferase activity of cells that were co-transfected with constructed luciferase reporters (ELAVL1 WT or ELAVL1 MUT), and miR-133a mimics or negative control. E and F The expression of ELAVL1, NLRP3, and cleaved caspase-1 in H/R cardiomyocytes by western blotting when transfected with miR-133a mimics or miR-133a inhibitor. G Cell viability was measured by CCK8 kit in H/R cardiomyocytes that transfected with miR-133a mimics or miR-133a inhibitor. n = 3, data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the NC group

Fig. 6. Exosome miR-133a protects against MI/R injury in vivo. A and B Protein expression and quantitative analysis in rat heart tissues. C and D ELISA detected the IL-1β, IL-18 expression in rat serum. E Histopathology and cell death were analyzed by HE and Tunel staining. n = 3, data are shown as mean ± SD. *p < 0.05, **p < 0.01 vs. the sham group