Краткое описание

Ubiquitin-dependent proteasomal proteolysis is crucial in turnover of cardiomyocytes functional proteins (actin, myosin, ion channels at al), therefore investigation of cell death after ubiquitin (UBB) gene silencing using RNA interference and anoxia-reoxygenation (AR) modeling appears to be attractive. Cardiomyocytes were transfected by siRNA to ubiquitin gene using electroporation procedure then primary culture was treated by 30 min of anoxia and 60 min of reoxygenation. The number of living, necrotic and apoptotic cardiomyocytes was determined by fluorescence microscopy. The level of UBB and proteasome subunits β5 (PSMB5) and LMP2 (PSMB9) mRNA expression was estimated by real-time PCR. It was shown that UBB mRNA expression was extended in 2.1 times after AR modeling (P < 0.05). Small interference RNA injection in cell culture decreased ubiquitin expression in 2.4 times (P < 0.05), PSMB5 - in 1.3 (P >0.05) and PSMB9 - in 1.6 (P < 0.05) times respectively compared with control (scrambled siRNA introduction). At the same time, the level of living cardiomyocytes was decreased ((70.26 ± 1.54)%, Р < 0.05), and augmented level of necrotic ((25.92 ± 1.52)%, Р = 0.38), apoptotic cells ((4.32 ± 0.53)%, Р = 0.15) and cells with signs of autophagy ((38.2 ± 3.8)%, Р = 0.001). But ubiquitin silencing after AR (30 min/1 h) increased number of living cells at 3.7% and decreased level of necrotic cells at 4.7% and no altered of apoptotic and autophagic cells populations. Obtained data testify that ubiquitin gene silencing, mRNA expression of which augmented during AR, induces necrotic and autophagic death of intact neonatal cardiomyocytes in culture, but enhances AR resistance of these cells to some extent.