Platelet-derived exosomes from septic shock patients induce myocardial dysfunction

Abstract

Introduction: Mechanisms underlying inotropic failure in septic shock are incompletely understood. We previously identified the presence of exosomes in the plasma of septic shock patients. These exosomes are released mainly by platelets, produce superoxide, and induce apoptosis in vascular cells by a redox-dependent pathway. We hypothesized that circulating platelet-derived exosomes could contribute to inotropic dysfunction of sepsis.

Methods: We collected blood samples from 55 patients with septic shock and 12 healthy volunteers for exosome separation. Exosomes from septic patients and healthy individuals were investigated concerning their myocardial depressant effect in isolated heart and papillary muscle preparations.

Results: Exosomes from the plasma of septic patients significantly decreased positive and negative derivatives of left ventricular pressure in isolated rabbit hearts or developed tension and its first positive derivative in papillary muscles. Exosomes from healthy individuals decreased these variables non-significantly. In hearts from rabbits previously exposed to endotoxin, septic exosomes decreased positive and negative derivatives of ventricular pressure. This negative inotropic effect was fully reversible upon withdrawal of exosomes. Nitric oxide (NO) production from exosomes derived from septic shock patients was demonstrated by fluorescence. Also, there was an increase in myocardial nitrate content after exposure to septic exosomes.

Conclusion: Circulating platelet-derived exosomes from septic patients induced myocardial dysfunction in isolated heart and papillary muscle preparations, a phenomenon enhanced by previous in vivo exposure to lipopolysaccharide. The generation of NO by septic exosomes and the increased myocardial nitrate content after incubation with exosomes from septic patients suggest an NO-dependent mechanism that may contribute to myocardial dysfunction of sepsis.

Figure 1

Exosomes from patients with septic shock. Electron micrograph of exosomes isolated from the plasma of patients with sepsis, showing round particles with diameters ranging from 50 to 150 nm. Magnification, × 41,000.

Figure 2

Effect of exosomes on left ventricular pressure in isolated rabbit heart preparations. Time course of positive (a) and negative (b) derivatives of left ventricular pressure after infusion of exosomes from septic patients and healthy volunteers. Data are mean ± standard error of the mean of nine experiments for exosomes derived from septic patients and five experiments for exosomes from healthy individuals. *p < 0.05 versus baseline; ^#p < 0.05 versus 20 minutes (analysis of variance two-way, Tukey test). +dP/dt_max, maximal positive derivative of left ventricular pressure; -dP/dt_max, maximal negative derivative of left ventricular pressure.

Figure 3

Effect of reactive oxygen species (ROS) inhibitors on exosome-induced inhibition of left ventricular pressure. Effect of ROS inhibitors on exosome-induced inhibition of maximal positive (a) and negative (b) derivatives of ventricular pressure. Data are mean ± standard error of the mean of nine experiments for exosomes from septic patients, four experiments for septic exosomes + L-monomethyl-arginine (LNMMA), five experiments for septic exosomes + n-acetylcysteine (NAC), four experiments for septic exosomes + dyphenyleneiodonium (DPI), three experiments for septic exosomes + apocynin (APO), and three experiments for septic exosomes + indomethacin (INDO). *p < 0.05 versus septic; (analysis of variance two-way, Tukey test). +dP/dt_max, maximal positive derivative of left ventricular pressure; -dP/dt_max, maximal negative derivative of left ventricular pressure.

Figure 4

Amplification of the effects of exosomes on left ventricular contractility upon previous exposure to lipopolysaccharide (LPS). Effect of exosomes on percentage of maximal positive (a) and negative (b) derivatives of left ventricular pressure in endotoxemic hearts. Data are mean ± standard error of the mean of experiments with exosomes from septic patients on normal hearts (septic ex, n = 9), exosomes from healthy individuals on normal hearts (healthy ex, n = 5), and exosomes from septic patients in endotoxemic hearts (septic ex + LPS, n = 4). *p < 0.05 versus baseline; ^#p < 0.05 versus septic ex; ^§p < 0.05 versus healthy ex (analysis of variance two-way, Tukey test). +dP/dt_max, maximal positive derivative of left ventricular pressure; -dP/dt_max, maximal negative derivative of left ventricular pressure.

Figure 5

Effect of exosomes in isolated rat papillary muscle preparations. Effect of exosomes on developed tension (a) and its positive derivative (+dT/dt) (b) in isolated rat papillary muscle preparations. Data are mean ± standard error of the mean of eight experiments for papillary muscles before and after incubation with exosomes derived from septic patients (septic pre and post) and four experiments for papillary muscles before and after incubation with exosomes derived from healthy individuals (healthy pre and post). *p < 0.05 versus septic pre (paired t test).

Figure 6

Exosomes exhibit intrinsic nitric oxide (NO) production and induce myocardial NO production. (a) 4,5-diaminofluorescein-2 (DAF-2) fluorescence (an index of NO production) by exosomes from septic shock patients and healthy volunteers. Data are mean ± standard error of the mean (SEM) of six experiments with exosomes from healthy volunteers and septic patients. *p < 0.05 versus healthy; ^§p < 0.05 versus baseline healthy; ^#p < 0.05 versus baseline septic (one-way analysis of variance, Student-Newman-Keuls test). Black bars: baseline septic and healthy exosomes; gray bars: septic and healthy exosomes + superoxide dismutase (SOD) (250 IU/mL); white bars: septic and healthy exosomes + N(G)-nitro-L-arginine methyl ester (L-NAME) (100 μM). (b) Myocardial nitrate content of hearts exposed for 45 minutes to exosomes from septic patients or healthy volunteers. Data are mean ± SEM of four experiments for exosomes of septic patients and healthy volunteers. *p < 0.05 versus healthy (t test). RFU, relative fluorescence unit.