Plasma Exosomes at the Late Phase of Remote Ischemic Pre-conditioning Attenuate Myocardial Ischemia-Reperfusion Injury Through Transferring miR-126a-3p

Abstract

Background: Remote ischemic pre-conditioning (RIPC) alleviated the myocardial ischemia-reperfusion injury, yet the underlying mechanisms remain to be fully elucidated, especially at the late phase. Searching a key component as a transfer carrier may provide a novel insight into RIPC-mediated cardioprotection in the condition of myocardial ischemia-reperfusion. Objective: To investigate the cardioprotective effect of plasma exosomes at the late phase of RIPC and its potential signaling pathways involved. Methods and Results: Exosomes were isolated from the plasma of rats 48 h after the RIPC or control protocol. Although the total plasma exosomes level had no significant change at the late phase of RIPC (RIPC-exosome) compared with the control exosomes (Control-exosome), the RIPC-exosome afforded remarkable protection against myocardial ischemia-reperfusion (MI/R) injury in rats and hypoxia-reoxygenation (H/R) injury in cells. The miRNA array revealed significant enrichment of miR-126a-3p in RIPC-exosome. Importantly, both miR-126a-3p inhibitor and antagonist significantly blunted the cardioprotection of RIPC-exosome in H/R cells and MI/R rats, respectively, while miR-126a-3p mimic and agomir showed significant cardioprotection against H/R injury in cells and MI/R injury in rats. Mechanistically, RIPC-exosome, especially exosomal miR-126a-3p, activated the reperfusion injury salvage kinase (RISK) pathway by enhancing the phosphorylation of Akt and Erk1/2, and simultaneously inhibited Caspase-3 mediated apoptotic signaling. Conclusions: Our findings reveal a novel myocardial protective mechanism that plasma exosomes at the late phase of RIPC attenuate myocardial ischemia-reperfusion injury via exosomal miR-126a-3p.

Keywords: cardiomyocyte apoptosis; cardioprotection; exosomes; miR-126a-3p; microRNA; myocardial ischemia-reperfusion; remote ischemic pre-conditioning.

Figure 1

Identification and functional verification of plasma exosomes at the late phase of RIPC in vitro. (A) Establishment of the late-phase RIPC model in rats. (B) The process to obtain the plasma exosomes from rats with or without the late-phase RIPC protocol. (C) Representative electron micrograph of isolated plasma exosomes. Scale bar: 100 nm. (D) Representative results of NTA (nanoparticle tracking analysis) demonstrating size distribution and concentration in Control-exosomes (C-exo) and RIPC-exosomes (R-exo). (E) The average concentration in Control-exosomes and RIPC-exosomes (n = 4). (F) Mode size in Control-exosomes and RIPC-exosomes (n = 4). (G) Mean size in Control-exosomes and RIPC-exosomes (n = 4). (H) Representative images of Western blot (top) and quantified data (bottom) in exosomal marker proteins CD81 in Control-exosomes and RIPC-exosomes (n = 4). (I) Representative images of Western blot (top) and quantified data (bottom) in exosomal marker proteins CD9 in Control-exosomes and RIPC-exosomes (n = 4). (J) Representative images of H9C2 cells that were incubated with the presence (H9C2-EXO) or absence (H9C2) of Dio-labeled exosomes (green). Cardiomyocyte nuclei were stained with DAPI (blue). Scale bar: 25 μm. (K) The data of cell viability showed that RIPC-exosomes improved cell viability with 24 h co-incubation and following H/R injury. (L) The data and representative images of cell apoptosis by flow cytometric analysis showed that RIPC-exosomes decreased cell apoptosis with 24 h co-incubation and following H/R injury (n = 3). RIPC, remote ischemic pre-conditioning; R-exo, exosomes isolated from the plasma of the late-phase RIPC rats; C-exo, exosomes isolated from the plasma of the control rats; H/R, Hypoxia/Reoxygenation (24/6 h). *p < 0.05 and ^nsp > 0.05.

Figure 2

Plasma exosomes at the late phase of RIPC exerted cardioprotection against MI/R injury in vivo. (A) Representative echocardiographic images of rats before the intervention (pre-intervention) and after the reperfusion (post-reperfusion) in each group of rats. (B) Left ventricular ejection fraction (LVEF) of rats in pre-intervention (n = 8). (C) ΔLVEF (the difference between the LVEF in pre-intervention and that in post-reperfusion) in each group. RIPC-exosome injected into rats for 24 h before MI/R improved cardiac function as evidence by significantly narrowed the ΔLVEF. (D) Quantified data and presentative photographs of heart sections. RIPC-exosome decreased infarct size in rats subjected to MI/R (n = 8). INF/AAR, infarct size as a percentage of myocardial area at risk. (E) Representative images and quantified data of TUNEL assay in each group showed that RIPC-exosome decreased myocardial apoptosis in rats subjected to MI/R. Myocardial apoptosis in rats determined by a percent of TUNEL-positive nuclei (green)/total nuclei (blue) (n = 4). Scale bar: 50 μm. MI/R, myocardial ischemia/reperfusion (30/180 min). R-exo, exosomes isolated from the plasma of the late-phase RIPC rats. C-exo, exosomes isolated from the plasma of the control rats. R-fep, the supernatant collected after the first ultracentrifugation from the plasma of the late-phase RIPC rats. C-fep, the supernatant collected after the first ultracentrifugation from the plasma of the control rats. *p < 0.05, ^nsp > 0.05.

Figure 3

miR-126a-3p was essential in the cardioprotection provided by plasma exosomes at the late phase of RIPC. (A) Venn diagram showing the numbers of overlapping and unique miRNAs detected in Control-exosome (C-exo) and RIPC-exosome (R-exo) (n =3). (B) The expression level of miRNAs in Control-exosome and RIPC-exosome. Yellow dots represented up-regulated miRNAs in RIPC-exosome and blue dots represented up-regulated miRNAs in Control-exosome (fold change >2 and p < 0.05, n =3). (C) Heat map representing the miRNA profiling assays between Control-exosome and RIPC-exosome (fold change >2 and p < 0.05, n =3). (D) qRT-PCR analysis of the six differentially expressed miRNAs in Control-exosome and RIPC-exosome. Data are normalized to cel-miR-39 (n =3). (E) The qRT-PCR analysis of miR-126a-3p level in H9C2 cells treated with inhibitor negative control (NC), R-exo + NC, or R-exo + miR-126a-3p inhibitor followed by H/R. Data were presented as a fold change of the normoxia. U6 was used as an internal control (n = 3). (F) The data of cell viability showed that inhibition of miR-126a-3p attenuated the protective effects of RIPC-exosome in H9C2 cells subjected to H/R (n = 3). (G) The data and representative images of apoptosis of H9C2 cells subjected to H/R by flow cytometric analysis (n = 3). The protective effects of RIPC-exosome were eliminated by inhibition of miR-126a-3p. (H) The data of cell viability showed that the protective effects of RIPC-exosomal miR-126a-3p were reduced by a miR-126a-3p inhibitor in primary cardiomyocytes following H/R injury (n =3). (I) The data and representative images of apoptosis of primary cardiomyocytes subjected to H/R by flow cytometric analysis (n = 3). The anti-apoptosis effects of RIPC-exosomal miR-126a-3p were eliminated by inhibition of miR-126a-3p (n =3). (J) Representative echocardiographic images of rats in pre-intervention and post-reperfusion in each group of rats (n = 5). (K) Left ventricular ejection fraction (LVEF) of rats in pre-intervention. (L) ΔLVEF in each group showed that miR-126a-3p antagomir blunted the protective effects of RIPC-exosome in cardiac function. (M) Representative photographs of heart sections and quantified data demonstrated that miR-126a-3p antagomir observably attenuated the protective effects of RIPC-exosome in myocardial infarct size (n = 5). (N) Representative images and quantified data of TUNEL assay in each group demonstrated that miR-126a-3p antagomir observably blunted the protective effects of RIPC-exosome in myocardial apoptosis (n = 4). Scale bar: 50 μm. R-exo, exosomes isolated from the plasma of the late-phase RIPC rats. C-exo, exosomes isolated from the plasma of the control rats. H/R, Hypoxia/Reoxygenation (24/6 h). MI/R, myocardial ischemia/reperfusion (30/180 min). *p < 0.05.

Figure 4

miR-126a-3p was an important molecule in cardioprotection. (A) The qRT-PCR analysis of miR-126a-3p level in H9C2 cells transfected with miR-126a-3p mimic or negative control (mimic-NC) for 24 h followed by H/R. Data were presented as a fold change of the normoxia. U6 was used as an internal control (n = 3). (B) The increasing level of miR-126a-3p improved cell viability in H9C2 cells subjected to H/R (n = 3). (C) Flow cytometric data and representative images of apoptosis in H9C2 cells subjected to H/R (n = 3). The increasing level of miR-126a-3p decreased cell apoptosis in H9C2 cells subjected to H/R. (D) Cell viability in primary cardiomyocytes transfected with a miR-126a-3p mimic or mimic-NC followed by H/R injury (n =3). (E) Flow cytometric data and representative images of the anti-apoptosis effect of miR-126a-3p in primary cardiomyocytes treated with a miR-126a-3p mimic or mimic-NC followed by H/R injury (n =3). (F) Representative echocardiographic images of rats in pre-intervention and post-reperfusion in each group of rats (n = 5). (G) Left ventricular ejection fraction (LVEF) of rats in pre-intervention. (H) ΔLVEF in each group showed that miR-126a-3p agomir improved cardiac function. (I) Representative photographs of heart sections and quantified data demonstrated that miR-126a-3p agomir reduced myocardial infarct size (n = 5). (J) Representative images and quantified data of TUNEL assay in each group demonstrated that miR-126a-3p agomir reduced myocardial apoptosis (n = 4). Scale bar: 50 μm. (K) Cell viability in H9C2 cells treated with a miR-126a-3p inhibitor or inhibitor-NC followed by H/R (n = 3). (L) Flow cytometric data and representative images of apoptosis in H9C2 cells treated with a miR-126a-3p inhibitor or inhibitor-NC followed by H/R (n = 3). H/R, Hypoxia/Reoxygenation (24/6 h). MI/R, myocardial ischemia/reperfusion (30/180 min). *p < 0.05.

Figure 5

The signaling pathways involved in plasma exosomes at the late phase of RIPC mediated effects. (A) Representative images of Western blot and quantified data in rats received R-exo, C-exo, R-fep, C-fep, or vehicle for 24 h followed by MI/R. (n = 3). (B) Representative images of Western blot and quantified data in H9C2 cells preincubated with R-exo, C-exo, or vehicle for 24 h followed by H/R (n = 3). GAPDH was used as a loading control. Quantified data showed phosphorylation level of Akt, expressed as the ratio of p-Akt to Akt; phosphorylation level of Erk1/2, expressed as the ratio of p-Erk1/2 to Erk1/2; the activation of Caspase-3 expressed as the ratio of cleaved Caspase-3 to Caspase-3. MI/R, myocardial ischemia/reperfusion (30/180 min). H/R, Hypoxia/Reoxygenation (24/6 h). R-exo, exosomes isolated from the plasma of the late-phase RIPC rats; C-exo, exosomes isolated from the plasma of the control rats; R-fep, the supernatant collected after the first ultracentrifugation from the plasma of the late-phase RIPC rats; C-fep, the supernatant collected after the first ultracentrifugation from the plasma of the control rats. *p < 0.05, ^nsp > 0.05.

Figure 6

The signaling pathways involved in plasma exosomes at the late phase of RIPC via miR-126-3p mediated effects. (A) Representative images of Western blot and quantified data in H9C2 cells treated with inhibitor-NC, R-exo + NC, or R-exo + miR-126a-3p inhibitor followed by H/R (n = 3). (B) Representative images of Western blot and quantified data in H9C2 cells transfected with miR-126a-3p mimics or mimic-NC for 24 h followed by H/R (n = 3). (C) Representative images of Western blot and quantified data in H9C2 cells transfected with miR-126a-3p inhibitors or negative control (inhibitor-NC) for 24 h followed by H/R (n = 3). GAPDH was used as a loading control. Quantified data showed phosphorylation level of Akt, expressed as the ratio of p-Akt to Akt; phosphorylation level of Erk1/2, expressed as the ratio of p-Erk1/2 to Erk1/2; the activation of Caspase-3 expressed as the ratio of cleaved Caspase-3 to Caspase-3. H/R, Hypoxia/Reoxygenation (24/6 h). R-exo, exosomes isolated from the plasma of the late-phase RIPC rats. *p < 0.05.

Figure 7

Proposed mechanisms by which plasma exosomes at the late phase of RIPC protect the heart against MI/R injury through transferring miR-126a-3p. Plasma exosomes could be used as a carrier to transfer the myocardial protective effect of RIPC at the late phase against MI/R injury, activate the RISK pathway by increasing the phosphorylation level of Akt, and Erk1/2, and inhibit the activation of apoptotic protein Caspase-3 by reducing the expression level of cleaved Caspase-3.