Myocardial infarction affects Cx43 content of extracellular vesicles secreted by cardiomyocytes

Figures

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  Figure 1.. Ischemia decreases secretion of Cx43 into circulating extracellular vesicles (EVs) in mice subjected to myocardial I/R injury. Left coronary artery ligation (60 min) was performed in mice, followed by reperfusion during 30 min (I/R 30′) or 4 h (I/R 4H). Sham-operated animals were used as controls (CT). (A) Non-reducing WB of circulating EVs (30 μg total protein/lane) from sham (cEVmouseCT), I/R 4H (cEVmouseI/R 4H), and I/R 30′ (cEVmouseI/R 30′). CD63 and CD9 were used as positive EV markers, Calnexin as a negative marker and Troponin T as a cardiomyocyte marker. Heart lysates were used as control. (B) Nanoparticle tracking analysis of mouse circulating EVs. (C) Representative transmission electron microscopy of mouse circulating EVs. (D) WB analysis of Cx43 in circulating EVs (30 μg total protein/lane, n = 6). GAPDH was used as a pan-EV marker. (E) WB analysis of Cx43 in intracardiac EVs (5 μg total protein/lane) from sham (iEVmouseCT), I/R 4H (iEVmouseI/R 4H), and I/R 30′ (iEVmouseI/R 30′; n = 6). Source data are available for this figure.

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  Figure S1.. Ischemia decreases secretion of Cx43 into circulating extracellular vesicles (EVs) from mice subjected to myocardial ischemia/reperfusion (I/R) injury and cardiomyocyte-derived EVs. (A) Total protein quantification of circulating EVs isolated from sham (cEVmouseCT), I/R 4H (cEVmouseI/R 4H) and I/R 30′ (cEVmouseI/R 30′) mice (n = 6). (B) Representative nanoparticle tracking analysis (NTA) of concentration and size distribution of intracardiac EVs obtained from sham (iEVmouseCT), or animals subjected to I/R for 4 h (iEVmouseI/R 4H) or 30 min (iEVmouseI/R 30′). (C) Representative transmission electron microscopy images of mouse-derived intracardiac EVs. (D) Total protein quantification of iEVmouseCT, iEVmouseI/R 4H, and iEVmouseI/R 30′ (n = 6). (E) Intracardiac EVs (5 μg total protein/lane) were isolated from Langendorff-perfused hearts, maintained in control perfusion or subjected to global myocardial ischemia by low flow (iEVrat ISCH). Non-reducing WB for the cardiac marker Troponin T and EV markers CD63 and CD81. (F) Representative transmission electron microscopy images of rat-derived intracardiac EVs. (G) Representative NTA of intracardiac EVs obtained from control (iEVrat CT)…

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  Figure 2.. Ischemia decreases sorting of Cx43 into cardiomyocyte-derived extracellular vesicles (EVs). (A) WB analysis of EV-Cx43 derived from HL-1 cells subjected to 30-min ischemia (ISCH), followed by 1 or 4 h of reperfusion (I/R). Flotillin-1 and GAPDH were used as pan-EV markers (n = 4). (B) WB analysis of EV-Cx43 derived from HL-1 cells subjected to 30 min ischemia and depleted or not of p62 (sip62; n = 4). Source data are available for this figure.

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  Figure S2.. Ischemia does not affect Cx43 secretion into small extracellular vesicles (EVs) derived by cardiac endothelial cells. (A) Representative transmission electron microscopy images of EVs (16,500g or 120,000g, as indicated) isolated from cardiac endothelial cells subjected or not to ischemia (ISCH). (B) Representative nanoparticle tracking analysis of concentration and size distribution of EVs derived from cardiac endothelial cells. (C) Non-reducing WB of cardiac endothelial EVs. CD63 and CD81 were used as canonical EV markers. The absence of Calnexin excludes contamination with cellular material. The presence of higher molecular bands (∼258 kD) of Cx43 denotes the presence of Cx43 hemichannels in EVs. (D) Levels of Cx43 in cardiac endothelial EVs derived from control or ischemic cells (n = 6). Analysis of the pan-EV markers Flotillin-1 and GAPDH was also performed.

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  Figure S3.. p62 regulates the balance between Cx43 degradation and extracellular vesicle (EV) secretion. (A) Representative nanoparticle tracking analysis (NTA) of concentration and size distribution of EVs obtained from HL-1 cells after siRNA-mediated knockdown of p62 (sip62) and/or subjected to ischemia (ISCH). (B) knockdown of p62 (sip62) was performed in HEK293Cx43+ cells for 48 h. Cells were cultured in EV-depleted medium for 8 h, followed by EV isolation. Levels of EV-Cx43 were evaluated by WB (n = 4). Analysis of the pan-EV markers Flotillin-1 and GAPDH was also performed. (C) Representative NTA of EVs derived from HEK293Cx43+ cells depleted or not of p62 (sip62). (D) HL-1 cells were transduced with shRNA targeting Atg7 (sh Atg7), or the empty lentiviral vector (CT) for 7 d, after which cells were incubated in EV-depleted medium for 8 h. WB analysis of EV-Cx43 levels was further performed (n = 4). (E) HEK293Cx43− cells were transfected with wild type Cx43 (Cx43WT) or Cx43 mutated in the residues S373 (Cx43S373A) or S368 (Cx43S368A) for 24 h. Cells were incubated in EV-depleted medium for 8 h, followed by WB analysis of EV-Cx43 levels (n = 4). (F) Representative NTA of concentration…

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  Figure 3.. Extracellular vesicle (EV)-Cx43 is phosphorylated and ubiquitinated. (A) Cx43 was immunoprecipitated (IP) from HEK293Cx43+-derived EVs. Cx43 phosphorylation was evaluated with phospho-Ser, phospho-Thr, and phospho-Tyr antibodies. (B) Phosphorylation of Cx43-S373 and Cx43-S368 was evaluated in HEK293Cx43+-derived EVs. (C) HEK293Cx43+ cells were treated with PMA or vehicle for 30 min in EV-depleted medium. EV-Cx43 levels were evaluated by WB (n = 4). (D) Cx43 ubiquitination evaluated after IP of Cx43 from HEK293Cx43+ cells. (E) HL-1 cells were treated with PMA or vehicle control for 30 min in EV-depleted medium. EV-Cx43 levels were assessed by WB (n = 4). (F) Cx43 ubiquitination evaluated after IP of Cx43 from HL-1 cells. (G) Cx43 ubiquitination evaluated after IP of Cx43 in HEK293Cx43+-derived EVs. (H) WB analysis of EV-Cx43 derived from HEK293Cx43+ cells knockdown for Tsg101 (siTsg101), incubated in EV-depleted medium for 8 h (n = 4). NP, non-phosphorylated Cx43; P2, phosphorylated Cx43. Source data are available for this figure.

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  Figure S4.. Cx43 found in extracellular vesicles (EVs) is of endosomal origin. (A) Ubiquitinated substrates from lysates of EVs derived from HEK293Cx43+ cells were precipitated using TUBEs. Following elution, samples were analyzed by WB using antibodies against Cx43 and ubiquitin. Cellular extracts were used as control. (B) Representative nanoparticle tracking analysis (NTA) of concentration and size distribution of EVCT and EVsiTsg101. (C) HEK293Cx43+ cells transfected with GFP or Rab5QLGFP for 24 h, were incubated in EV-depleted medium for 8 h. Cx43 levels in EVs were evaluated by WB (n = 6). Analysis of the pan-EV markers Flotillin-1 and GAPDH was also performed. (D) Representative NTA of concentration and size distribution of EVGFP, EVGFP+PMA, EVRab5QL, and EVRab5QL+PMA. (E) HEK293Cx43+ cells transfected with Rab5QLGFP for 24 h were treated with PMA, where indicated, and immunostained for Cx43 (red) and Tsg101 (white). Nuclei were stained with DAPI. Scale bars 5 μm. Quantification of the number of Rab5QLGFP endosomes filled with Cx43 is depicted on graph (n = 4). (F) HL-1 cells transfected with Rab5QLGFP for 24 h were treated with PMA, where indicated, and immunostained for Cx43 (red).…

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  Figure 4.. Ubiquitination promotes secretion of extracellular vesicle (EV)-Cx43. (A) WB analysis of EV-Cx43 derived from HEK293Cx43+ cells knockdown for Nedd4 (siNedd4), incubated in EV-depleted medium for 8 h (n = 5). (B) Cx43 ubiquitination in HEK293Cx43+ cells evaluated after immunoprecipitation of Cx43. (C) WB analysis of EV-Cx43 derived from HEK293Cx43+ cells knockdown for AMSH (si AMSH) or USP8 (siUSP8), incubated in EV-depleted medium for 8 h (n = 7). Source data are available for this figure.

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  Figure S5.. Ubiquitination promotes secretion of Cx43 into extracellular vesicles (EVs). (A) HEK293Cx43+ cells were either treated with Heclin or vehicle control for 2 h in EV-depleted medium. EVs were isolated, after which the levels of Cx43 were analyzed by WB (n = 4). Analysis of the pan-EV markers Flotillin-1 and GAPDH was also performed. (B) Representative nanoparticle tracking analysis (NTA) of EVs derived from cells treated or not with Heclin. (C) Representative NTA of concentration and size distribution of EVCT and EVsiNedd4. (D) Immunoprecipitation of Cx43 was performed in HEK293Cx43+ cells treated or not with Heclin for 2 h. Cx43 ubiquitination was evaluated by WB. (E) Cx43 was immunoprecipitation from HEK293Cx43− cells transfected with either Cx43WT or ubiquitination-defective Cx43 mutated in Y265 and Y286 resides (Cx43Y265/286A). Ubiquitination levels of Cx43 were analyzed by WB. (F) HEK293Cx43− cells were transfected with Cx43WT or Cx43Y265/286A for 24 h, followed by immunostaining for Cx43 (green). Nuclei were stained with DAPI. Scale bars 10 μm. (G) HEK293Cx43− cells transfected with Cx43WT or Cx43Y265/286A were incubated in EV-depleted medium for 8 h. EV-Cx43 levels were…

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  Figure S6.. Cx43-containing vesicles can be retained in the ECM. (A) Representative transmission electron microscopy image of ECM-bound extracellular vesicles (EVs) purified by differential ultracentrifugation. (B) WB analysis of ECM-bound EVs purified by differential ultracentrifugation and total ECM fractions. (C) EVs were isolated from conditioned medium of cells expressing or not Cx43 (EVCx43+ or EVCx43−) – EV input (10 μg total protein). Isolated vesicles were incubated with collagenase/DNAse for 1 h, at 37°C, followed by WB analysis. (D) Levels of Cx43 were determined in EVs derived from HEK293Cx43+ cells, either subjected to ischemia (ISCH) for 30 min or treated with Heclin for 2 h (n = 4). (E) Levels of Cx43 in total ECM fractions from HEK293Cx43+ cells, either subjected to ischemia (ISCH) for 30 min or treated with Heclin for 2 h (n = 4). SART1 was used to determine cellular contamination. (F) Representative transmission electron microscopy analysis of synthetic collagen fibers before (up) and after incubation with purified EVs (bottom) derived from HEK293Cx43+ cells. (G) Purified EVs derived from HEK293Cx43+ cells subjected to 30 min of ischemia (ISCH) or treated with Heclin for 2…

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  Figure 5.. I/R impacts retention of extracellular vesicle (EV)-Cx43 in the ECM. (A) Non-reducing WB of HEK293Cx43+-derived ECM (10 μg total protein/lane). CD63, CD81, CD9, Tsg101, Flotillin-1, and GAPDH was used as EV markers. p53 was used to determine cellular contamination. (B) Representative transmission electron microscopy of HEK293Cx43+-derived ECM. (C) Levels of Cx43 in ECM (10 μg total protein/lane) from HL-1 cells subjected to 30 min ischemia (ISCH), 1 h or 4 h of reperfusion (I/R; n = 4). (D) Non-reducing WB of heart-derived ECM (10 μg total protein/lane) from sham (ECMmouseCT), I/R 4H (ECMmouseI/R 4H), and I/R 30′ (ECMmouseI/R 30′). (E) WB analysis of Cx43 in heart-derived ECM (10 μg total protein/lane; n = 6). Source data are available for this figure.

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  Figure S7.. Cx43 levels decrease in extracellular vesicles (EVs) secreted and bound to the ECM during ischemia. (A) Non-reducing WB of canonical EV markers–CD63, CD81, Tsg101, Flotillin-1, and GAPDH–in total ECM fractions isolated from HL-1 cells (10 μg of protein/sample). (B) Non-reducing WB of canonical EV markers in total ECM fractions isolated from mixed primary cultures of cardiomyocytes and fibroblasts (CM/FB; 10 μg of protein/sample). (C) Levels of Cx43 were evaluated in EVs derived from mixed primary cultures of CM/FB subjected to 30 min of ischemia (ISCH), followed by 1 or 4 h of reperfusion (I/R; n = 3). (D) WB analysis of Cx43 in total ECM fractions (10 μg total protein/lane) from mixed primary cultures of cardiomyocytes and fibroblasts subjected to ischemia (ISCH), followed by 1 or 4 h of reperfusion (I/R; n = 4). p53 was used to determine cellular contamination. (E) Mice hearts were harvested and decellularized by treatment with 0.2% SDS. ECM fractions derived from decellularized hearts were analyzed by scanning electron microscopy, transmission electron microscopy and Masson’s trichrome staining. (F) Total protein quantification of heart-derived ECM from sham (ECMmouseCT), I/R…