Background. The polymorphonuclear neutrophils (PMN) are very important type of cells that participate in the innate non-specific organisms’ defense. These cells fulfill phagocytosis, regulation of the inflammation and can strongly facilitate the secondary alteration of tissues due to reactive oxygen specious and proteolytic enzymes production. The specific cell death phenomenon has been described for PMN and named neutrophil extracellular traps (NET) formation or NETosis [1]. During this process the not fragmented DNA releases from the PMN forming the net and containing proteolytic enzymes. This structure passively catch and destroy different pathogens but also participates in different pathological processes such as thrombosis [2], autoimmune disorders [3] and tissue damage during sterile inflammation such as myocardial infarction [4, 5]. Protein kinase C and some autophagy-related proteins were shown as the main regulators of this process [6]. The ubiquitin-dependent proteasomal proteolysis is very important regulator of all cellular activities. It participates in apoptosis and autophagy regulation in all cell types including cardiomyocytes and PMN [7]. But the role of proteasomal proteolysis in NETs formation now is not investigated as well as the role of NETs formation in myocardial damage during hypoxia-reoxygenation.

The purpose of the study is to investigate the influence of NETs formation on cardiomyocytes damage in coculture and the possible effect of the proteasomal proteolysis inhibition on the NETs formation and cardiomyocytes damage.

Methods. PMN were isolated from rat blood using Percoll gradient. Isolated PMN were activated to form NETs by phorbol 12-myristate 13-acetate (PMA, 20nM). NETs were detected with use of fluorescence microscopy (Hoechst staining) and by measure of DNA concentration in cultivation media. Proteasomal inhibitor (clasto-Lactacystin β-lactone, 100nM) was added to PMN before activation. All three proteasomal activities were measured in PMN. Rat neonatal cardiomyocytes were cultivated 24 hours after isolation and then subjected to hypoxia-reoxygenation. Also activated PMN and PMN treated with the proteasomal inhibitor were added to cardiomyocytes culture with or without hypoxia-reoxygenation modeling. Apoptotic cell death as well as necrosis of cardiomyocytes were detected using fluorescence microscopy (Hoechst, and propidium iodide staining).

Results. In the isolated PMN the percentage of NETs after PMA application was 70,8 ± 7,2 % and proteasomal inhibitor decreased this amount to 4,7 ± 0,9 %. At the same time the apoptotic program in PMN was activated. In the culture of rat neonatal cardiomyocytes, the proteasomal inhibitor application  increased the number of apoptotic cardiomyocytes (in 1.93 and 2.7 times correspondingly, p < 0.01 comparing to control). The modeling of hypoxia-reoxygenation in the  culture of rat neonatal cardiomyocytes increased the amount of necrotic cells, but the treatment with the proteasomal inhibitor decreased this numberin 1.86 times (p < 0.05 comparing to hypoxia-reoxygenation without proteasomal inhibitor). The activated PMN in coculture with isolated cardiomyocytes leads to increase of necrotic cardiomyocytes amount up to 33,1 ± 12,9 % and corresponding decrease of living cardiomyocytes amount. The number of living cardiomyocytes in coculture with PMN treated with both PMA and proteasomal inhibitor was 76,6 ± 13,3 % (P<0.05)  and necrotic – 23,4 ± 13,3 % (P<0.05). PMA itself did not lead to significant changes in the amount of living, necrotic and apoptotic cardiomyocytes.

Conclusion. The data obtained show that the formation of NET by PMN directly damage cardiomyocytes in coculture. The proteasome participates in NETs formation and proteasomal inhibitor leads to block of NETs programme realization. In coculture with cardiomyocytes during anoxia-reoxygenation this effect prevents cardiac cell death induced by activated polymorphic mononuclear neutrophils.