Identification of Connexin 26 on Extracellular Vesicles from Human Cardiomyocytes and Plasma: Novel Insights into miRNA Loading and Oxidative Injury

Figures

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  Figure 1. Characterization of hiPSC-CMs and Cx26 expression in hiPSC-CM derived EVs. (A) hiPSC-CMs at light inverted microscopy. (B,C) Representative confocal images of cardiomyocytes derived from hiPSCs cultured in chamber slides and subjected to double immunofluorescence: (B) green immunopositive reaction for sarcomeric α-actinin and no visible red immunonegative reaction for Cx32, (C) green immunopositive reaction for Cx43 and red immunopositive reaction for Cx26; nucleus (DAPI, blue). Image in (C) was acquired in one Z-plane (8th of 30). (D) Representative TEM image of Cx26 immunogold analysis on EVs from hiPSC-CMs, performed with 10 nm gold particles conjugated to anti-rabbit secondary antibody. Arrows point to Cx26 immunopositive EVs.

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  Figure 2. Cx26 expression in human cardiac-derived plasma EVs. (A) Representative TEM images with 10 nm immunogold for Cx26 on EVs from frozen human plasma of donors 1–3 (plus unfrozen donor 1 sample, 1 bis). (B) Representative Western blot of EVs from human plasma of donors 1–4 showing Cx26 with flotillin 2 as EV marker (n = 2) and cTNT as cardiomyocyte marker (n = 1). (C) Semi-quantitative analysis of Cx26 levels in EVs from donors 2 and 3 (n = 2), * p < 0.05. (D) Representative double immunogold TEM images (10 nm gold for Cx26, 20 nm gold for cTNT) on EVs from donor 1; arrows indicate EVs positive for both Cx26 and cTNT; black arrowhead points to positive EVs only for Cx26 while the red ones only for cTNT. (E) cTnT blot of murine cardiac lysate. (F) Western blotting of EVs from human plasma of donors 1–4 probed with secondary antibodies only, anti-rabbit (left) or anti-mouse (right).

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  Figure 3. Characterization of dH9c2 cardiomyocytes under normal and oxidative stress conditions. Representative images of double immunofluorescence for cardiac muscle isoform of MYL2 ((A), green), sarcomeric α-actinin ((B), green) and Cx43 ((A,B), red). (C–F) Cell injury evaluation induced by H2O2 treatment. Representative images of immunofluorescence for 4HNE in control ((C), red) and H2O2 treated cells ((E), red), and CASP-3 in control ((D), green) and H2O2 treated cells ((F), green). In (D,F) the nuclei are marked in blue with DAPI. The brightness and contrast of the magnified images in the squares were increased in the same way for all enlargements, in order to facilitate the identification of the immunofluorescence reaction.

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  Figure 4. Characterization and sizing of EVs derived from dH9c2 cardiomyocytes. (A) a screenshot of video from NanoSight LM10 showing EVs. (B,C) Concentration of EVs derived from control or H2O2 treated cells: (B) total EVs, n = 3 or (C) exosomes, n = 3, * p < 0.05. (D,E) Characterization for the size and the number of EVs derived from control (D) and H2O2-treated (E) cells. (F) Representative TEM images of EVs from treated cells. Arrows point to aggregates of four or more EVs.

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  Figure 5. Cx26 expression in EVs derived from dH9c2 cardiomyocytes under normal and oxidative stress conditions. (A) Western blotting for Cx26 and CD63 of EVs from not treated (CNTRL) or H2O2-treated, under oxidative stress, cells. The upper panel shows a representative immunoblotting of EV lysates, while the lower panel shows the semi-quantitative analysis from six independent blotting experiments. * p < 0.05 vs. control. (B) TEM-Immunogold analysis of EVs from CNTRL and H2O2-treated cells. The upper panel shows representative images of EVs immunopositive for Cx26, where the aggregation state of the EVs, which frequently occurs, is particularly evident in the images of the control group. The lower panel presents the relative semi-quantitative analysis expressed as the number of colloidal gold particles for EV or for EV area. **** p < 0.0001 vs. control.

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  Figure 6. Unsilenced WT and Cx26-silenced H9c2 cells release EVs with different miRNAs cargo. (A) Representative images showing Cx26 and GAPDH red immunopositive staining in scrambled siRNA-silenced cells (WT, top), and immunonegative staining in cells treated with related siRNA (KD, bottom). (B) RT-PCR analysis. Real-time PCR results for Cx26 mRNA (threshold cycles, Ct values, and corresponding melting peaks [upper panels]) and RPL13a mRNA (threshold cycles, Ct values, and corresponding melting peaks [lower panels]) in WT (red) and Cx26-KD (green) cells. (C) Relative expression levels of miR-1, miR-30a, and miR-280a carried by EVs released by WT cells (n = 3, black bar) or Cx26-KD cells (n = 8, white bar). (D) Representative micrographs of immunogold for Cx26 in EVs from WT and Cx26-KD cells. Red arrows point to gold particles on single or grouped EVs from WT cells. Arrowhead points to unspecific gold particles non-bound to EVs from Cx26-KD cells.