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Objectives: Myocardial infarction induces neutrophil activation and the release of neutrophil extracellular traps (NETs) which are thought to increase the inflammatory response. NETs are composed of decondensed chromatin decorated with granule- and cytosol-derived proteases. Despite their increased abundance at the culprit lesion site in ST-elevation myocardial infarction (STEMI) and within the infarcted myocardium, their contribution to cardiomyocyte damage is insufficiently understood. Here, we studied the impact of NETs on the viability of murine and human iPS-cells derived cardiomyocytes in vitro.
Methods: Human circulating neutrophils and murine bone marrow-derived neutrophils were stimulated in vitro with phorbol 12-myristate 13-acetate (PMA) to induce NETs formation. NETs were purified from the supernatant of stimulated cells and stored until use. Human iPS-cells derived cardiomyocytes (iPS-CM) and murine neonatal cardiomyocytes (nCM) were incubated with 1000 ng/ml or 2000 ng/ml NETs for up to 24 hours. As a control, cells were additionally incubated with calf thymus DNA. After 8 h, intracellular reactive oxygen species (ROS) were quantified and cleaved caspase-3, an executor of apoptosis, was detected in nCM by immunofluorescence. After 24 h, the amount of lactate dehydrogenase (LDH) was quantified in culture supernatants and metabolic activity was assessed by MTS assay. Moreover, the percentage of TUNEL-positive cells displaying fragmented DNA was determined by immunofluorescence.
Results: NETs treatment increased intracellular ROS formation and the abundance of TUNEL-positive human and murine cardiomyocytes in a concentration-dependent manner. Moreover, enhanced LDH release from damaged iPS-CM has been observed, although the metabolic activity of cells remained unchanged. Similarly, nCM displayed increased caspase-3 activation after exposure to NETs but no alterations in metabolic activity. Importantly, purified DNA did not affect cardiomyocyte viability indicating that the detrimental effects of NETs are rather mediated by NETs-associated proteases. On the other hand, sustained metabolic activity in cardiomyocytes might indicate upregulation of compensatory mechanisms in order to maintain energy supply.
Conclusion: Our findings indicate that NETs exert cytotoxic effects on cultured human and murine cardiomyocytes through mechanisms involving LDH leakage and DNA fragmentation. Thus, therapeutic targeting of NETs seems a promising strategy to protect cardiomyocytes from inflammation-related damage.
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