The Effect Of Vitamin E On Apoptosis And Hypertrophy Of Neonatal Rat Cardiomyocytes Inducded By Noradrenaline

Noradrenaline(NA) is the primary sympathetic neurotransmitter and endocrine hormone in vivo. The direct effects of NA on cardiac myocytes may contribute to both heart failure and pathologic remodeling, and may be associated with cellular hypertrophy, apoptosis, and increased levels of oxidative stress. There is increasing evidence that reactive oxygen species (ROS) are signaling molecules under subtoxic conditions and play an important role in cellular processes such as transcription factor activation, apoptosis, and cell growth. Vitamin E is the most effective natural free radical scavenger identified to date. Basic science, animal studies, epidemiological and observational studies have showed the benefit of vitamin E in the prevention of cardiovascular disease and slow the progression. But we are far from understanding the mechanisms of its physiological functions. Therefore, we hypothesize that ROS mediates the effects of NA on myocyte apoptosis and hypertrophy. Using the cultured neonatal rat cardiomyocytes(NRCs), we examined whether NA induced hypertrophy and apoptosis of the neonatal rat cardiomyocytes via an intracellcular signaling pathway involving ROS; We have also study the effects and effects of Vitamin E on NA-induced ROS production, apoptosis and hypertrophy of the cultured NRCs. Further elucidation of the mechanisms leading to cardiac hypertrophy and apoptosis may open new routes of molecular cytology to prevention and treatment of CHF. Our study comprehended three parts:PART Ⅰ :The fluorescent prob, DCFH-DA, was used for the assessment of NA-induced intracellular ROS formation in cultured NRCs. The changes incell fluorescence intensity were measured by flow cytometer(FCM) at different time and dose; We studied the effects of 3 -blocker on NA-induced increase of intracellular ROS formation; We examined whether vitamin E reduces increase of NA induced DCFH-DA fluorescence in cardiomyocytes.PART Ⅱ : Here we investigate the characteristics of NA-induced apoptosis and hypertrophy, and whether all these responds could be prevented by vitamin E pretreatment.Experiment one: The NRCs were cultured with NA(2μmol/L, 20 μ mol/L. 200 μ mol/L)in free-serum medium for 36h in order to determine the best concentration of NA inducing apoptosis. Under this condition , NRCs were co-cultured with three end concentration of vitamin E(5μ mol/L , 50 μ mol/L, 500 μmol/L) and the DNA ladder and percentage of TUNEL-positive myocytes were evaluated.Experiment two: The neonatal rat cardiomyocytes(NRCs) were cultured with NA(2 μ mol/L, 20 μ mol/L. 200μ mol/L)in free-serum medium for 36h in order to determine the best concentration of NA inducing hypertrophy, as reflected by increased in 3H- Leucine incorporation and cellular protein content. Under this condition , the effects of vitamin E (5 μ mol/L , 50 μ mol/L, 500 μ mol/L) on NA-induced hypertrophy were studied.PART Ⅲ :The NA-induced activation of NF- K B was examined by electrophoretic mobility shift assay (EMSA), and we also investigate the effect of vitamin E and β -bloker on NF- K B activity in NRCs.RESULTThe result of experiment part I showed that NA increased myocyte ROS production in a time- and concentration-dependent manner through adrenergic receptor pathway. Vitamin E inhibited the NA-induced increase of DCFH-DA fluorescence in cardiomyocytes in a concentration-dependentmanner.①Cultured NRCs were treated with NA(2 μ mol/L, 20 μ mol/L, 200 μ mol/L)and after Omin, 15min,30min of incubation, fluorescence intensity was measured by FCM. The fluorescence intensity of NRCs significantly increased as the concentration increased and time prolonged. ②Cultured NRCs were treated with NA(20 μ mol/L), NA-induced production of ROS was partially inhibited by prazosin and propranolol and completely inhibited by preincubation with both prazosin and proanolol in the cultured NRCs. ③NA at the concentration of 20 μ mol/L caused significant increase in intracellular ROS, these changes were significantly attenuated...