Methods: Before coronary artery ligation, rats were injected IP with a chemical inhibitor of neutral sphingomyelinase (nSMase) which is essential for the biogenesis and release of EVs. The number and size profile of plasma-derived EV was assessed by NTA analysis at baseline and 24hrs after MI. Multiple EV cytokine levels were simultaneously determined using enzyme-linked immunosorbent assay (ELISA)-based protein array technology. Heart global function was assessed by echocardiography and hemodynamic analysis performed at 7, 14 and 28 days after MI. Cytotoxic effects of circulating EV were evaluated ex-vivo in a Langedorff, system by measuring the level of cardiac troponin I (cTnI) in the perfusate. Mechanisms undergoing cytotoxic effects of EV derived from pro-inflammatory MF (MFM1) were studied in vitro into primary rat neonatal CM.
Results: The induction of MI and the consequent inflammation, dramatically increase the number of circulating EV carrying inflammatory cytokines such as IL1a, ILß and Rantes. Preventive inhibition nSMase significantly reduced the boost of inflammatory EV and cytokines in treated group as compared to control animals. The reduction of circulating EV post MI results in preserved LV ejection fraction at 7 and 28 days post-MI as compared to control group. Hemodynamic analysis confirmed functional recovery by displaying a higher velocity of LV relaxation and an improved contractility capacity in treated versus control group. The number of infiltrating CD68+ monocytes/macrophages in the infarct area was significantly reduced. Post-MI circulating EV induce cell death in adult CM when added to the perfusate of Langendorff, as assessed by the incresed level of cTnI into media. In vitro MFM1-EV activated nuclear translocation of NF-kB. Specific inhibition of TLR4 receptor activity abrogated NF-kB translocation and reduced cell death. Indicating that the axis TRL4-NF-kB is essential in EV-mediated CM death.
Conclusions: Systemic inhibition of EV release during the acute phase of MI preserves heart function in an animal model of LAD. These findings suggest detrimental effects of exosomes in the acute phase of MI.