Plasma exosomes generated by ischaemic preconditioning are cardioprotective in a rat heart failure model

Abstract

Background: Exosomes released into the plasma after brief cardiac ischaemia mediate subsequent cardioprotection. Whether donor exosomes can provide cardioprotection to recipients with chronic heart failure, which confers the highest perioperative risk, is unknown. We examined whether ischaemic preconditioning (IPC)-induced plasma exosomes exerted cardioprotection after their transfer from normal donors to post-infarcted failing hearts.

Methods: Plasma exosomes were obtained from adult rats after IPC or sham. An exosome inhibitor GW4869 was administrated before IPC in an in vivo model of ischaemia/reperfusion (I/R) injury in normal rats. The IPC exosomes or control exosomes from normal donor rats were perfused to the normal or post-infarcted failing rat hearts before ischaemia in Langendorff perfusion experiments. Infarct size, cardiac enzymes, cardiac function, and pro-survival kinases were quantified.

Results: The IPC stimulus increased the release of exosomes, whereas GW4869 inhibited the rise of plasma exosomes. Pre-treatment with GW4869 reversed IPC-mediated cardioprotection against in vivo I/R injury. In the Langendorff perfusion experiments, IPC exosomes from normal donor rats reduced mean infarct size from 41.05 (1.87)% to 31.43 (1.81)% and decreased lactate dehydrogenase activity in the post-infarcted failing rat hearts. IPC exosomes but not control exosomes activated pro-survival kinases in the heart tissues.

Conclusions: Ischaemic preconditioning-induced exosomes from normal rats can restore cardioprotection in heart failure after myocardial infarction, which is associated with activation of pro-survival protein kinases. These results suggest a potential perioperative therapeutic role for ischaemic preconditioning-induced exosomes.

Keywords: cardioprotection; exosome; heart failure; ischaemic preconditioning; pro-survival kinases.

Fig 1

IPC promotes the release of plasma exosomes in rats. (a) Schematic diagram illustrating the extraction and verification of exosome derived from normal rat plasma. (b) Representative transmission electron micrographs of exosomes extracted from rats in each group. Scale bar represents 100 nm. (c) Dynamic light scattering analysis of exosomes showing size distribution by volume. (d) Bar graph shows the exosome content in normal rat plasma at the end of ischaemic preconditioning (n=6 rats per group). (e) Representative bands of the exosomal protein CD63 and HSP70. (f) The relative levels of exosomal protein CD63 and HSP70 (n=4 per group). Data are presented as mean [standard error of the mean]. BCA, bicinchoninic acid assay; GW, GW4869; IPC, ischaemic preconditioning.

Fig 2

Effects of GW4869 on IPC-induced cardioprotection against in vivo I/R injury in normal rats. (a) Schematic diagram illustrating the in vivo experimental protocols. (b) Representative images of heart sections stained by triphenyltetrazolium chloride (TTC) and infarct size (IS) expressed as a percentage of area at risk (AAR) for each experimental group. The non-ischaemic area stained blue and AAR was brick red, whilst IS was white (n=6 rats per group). (c–d) The activities of cTnI and CK-MB in each group (n=6 rats per group). (e) Arrhythmia score was analysed in each group (n=6 rats per group). Data are presented as mean [standard error of the mean]. CK-MB, creatine phosphokinase-MB; cTnI, cardiac troponin I; GW, GW4869; IPC, ischaemic preconditioning; I/R, ischaemia/reperfusion.

Fig 3

IPC-derived exosomes exert cardioprotection against I/R injury in both normal and failing isolated rat hearts. (a) Schematic diagram illustrating the Langendorff experimental protocols. (b) Representative images of heart sections stained by triphenyltetrazolium chloride (TTC), and the infarct size (IS) is expressed as a percentage of area at risk (AAR) in normal isolated hearts. (c) Representative images of the failing heart sections stained by TTC, and the IS is expressed as a percentage of AAR in failing isolated heart (n=6). (d–e) The activity of lactate dehydrogenase (LDH) in coronary effluents was measured at baseline, 5 min, and 10 min after reperfusion (P5 and P10) in normal and failing isolated hearts. Data are presented as mean [standard error of the mean]. ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001; ∗∗∗∗P<0.0001. C-Exo, control exosome; I-Exo, IPC exosome; IPC, ischaemic preconditioning; I/R, ischaemia/reperfusion; ns, not significant.

Fig 4

Both ERK1/2 and Akt are activated by IPC exosomes in normal and failing isolated rat hearts. (a–b) Representative Western blot images and the relative expression levels of p-ERK1/2 normalised to the total ERK1/2 and GAPDH in (a) normal and (b) failing rat hearts (n=4 rats per group). (c–d) Representative Western blot images and the relative expression levels of p-Akt normalised to the total Akt and GAPDH in (c) normal and (d) failing rat hearts (n=4 rats per group). Data are presented as mean [standard error of the mean]. Akt, protein kinase B; C-Exo, control exosomes; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; I-Exo, IPC exosomes; I/R, ischaemia/reperfusion; Sham, sham operation.

Fig 5

Schematic shows Ischaemic preconditioning-derived exosomes from normal rats protect post-infarcted failing heart against ex vivo I/R injury by activating pro-survival kinases. Akt, protein kinase B; ERK, extracellular signal-regulated kinase; IPC, ischaemic preconditioning; I/R, ischaemia/reperfusion; P, phosphorylation.