Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C97
The Effect Of Hypoxia On NADPH Oxidase Isoforms Expression In Rat Neonatal Cardiomyocytes
M. Cherif1, M. T. Ghorbel1, M. Caputo1, S. Suleiman1, G. D. Angelini1
1. Bristol Heart Institute, Bristol, United Kingdom.
Myocardial ischaemia (hypoxia) and reperfusion (reoxygenation) induce injury partly by generating reactive oxygen species (ROS). NADPH oxidases (NOX) are an important source of ROS and there is evidence that NOX isoforms are important in cardiac pathophysiology (Cave et al., 2005, Sirker et al., 2007). In this study we investigated the effect of hypoxia on the expression and localisation of different NOX isoforms using an experimental model of cultured rat neonatal cardiomyocytes. Neonatal rat ventricular cardiomyocytes were obtained by digesting hearts from one-to three day old Wistar rats using trypsin (0.10%). Cardiomyocytes were cultured for 40 hours in medium containing 10% FBS and then for 24 hours in FBS free medium (Sala-Newby et al., 2003). Cells from three day old cultures were subjected to normoxia or hypoxia for 24 hours. Hypoxia (0.2% O2, 5%CO2) was induced by placing the cells in a ProOx hypoxic chamber where oxygen concentration was monitored. Changes of NOX isoforms were assessed by immunocytochemistry and western blotting. Hypoxia caused relocalisation of both NOX1 and NOX2 in cardiomyocytes. In normoxic cells, NOX1 was expressed in the cytoplasm, nucleus and peri- nucleus. However, during hypoxia NOX1 was largely expressed in the cytoplasm. NOX2 was located in the cytoplasm in normoxic cells whereas in hypoxic myocytes it was mainly localised to the membrane. NOX4 was located in the cytoplasm of both normoxic and hypoxic cells. Hypoxia did not affect protein content for NOX1 but significantly reduced protein expression for NOX2 and NOX4. We have used an experimental model of hypoxia in rat neonatal cardiomyocytes to demonstrate that hypoxia triggers changes in location and protein content of myocardial NOX. These changes are different for different isoforms. The significance of these changes in relation to ROS generation requires further investigation.
Where applicable, experiments conform with Society ethical requirements