[Effects of ubiquitin gene silencing in anoxia-reoxygenation of cultured cardiomyocytes].

Abstract

Ubiquitin-dependent proteasomal proteolysis is crucialin the 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, and 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 beta5 (PSMB5) and beta9 (PSMB9) mRNA expression was estimated by real-time PCR. It was shown that UBB mRNA expression was increased by 2.1 times after AR modelling (P < 0.05). Small interference RNA injection in cell culture decreased ubiquitin, PSMB5 and PSMB9 expression by 2.4 (P < 0.05), 1.3 (P > 0.05) and 1.6 (P < 0.05) times, respectively, compared with control (scrambled siRNA introduction). At the same time, the number of living cardiomyocytes decreased to 70.26 +/- 1.54%, P<0.05, and the level of necrotic cells, apoptotic cells and cells with signs of autophagy augmented by 25.92 +/- 1.52%, (P = 0.38), 4.32 +/- 0.53% (P = 0.15) and 38.2 +/- 3.81% (P = 0.001), respectively. Ubiquitin silencing after AR (30 min/l h) increased the number of living cells by 3.7% and decreased the number of necrotic cells by 4.7% and did not alter the apoptotic and autophagic cells populations. The data obtained indicate that ubiquitin gene silencing, mRNA expression of which augmented during AR, induces necrotic and autophagic death of intact neonatal cardiomyocytes in culture, but enhances the AR resistance of these cells to some extent.