Abstract
We have applied the highly sensitive chemiluminescence (CL) imaging technique to investigate the in situ ROS formation in cultured monolayers of rat H9c2 cardiomyocytes. Photon emission was detected via an innovative imaging system after incubation of H9c2 cells in culture with luminol and horseradish peroxidase (HRP), suggesting constitutive formation of ROS by the cardiomyocytes. Addition of benzo(a)pyrene-1,6-quinone (BPQ) to cultured H9c2 cells resulted in a 4-5-fold increase in the formation of ROS, as detected by the CL imaging. Both constitutive and BPQ-stimulated CL responses in cultured H9c2 cells were sustained for up to 1 hour. The CL responses were completely abolished in the presence of superoxide dismutase and catalase, suggesting the primary involvement of superoxide and hydrogen peroxide (H2O2). In contrast to BPQ-mediated redox cycling, blockage of mitochondrial electron transport chain by either antimycin A or rotenone exerted marginal effects on the ROS formation by cultured H9c2 cells. Upregulation of cellular antioxidants for detoxifying both superoxide and H2O2 by 3H-1,2-dithiole-3-thione resulted in marked inhibition of both constitutive and BPQ-augmented ROS formation in cultured H9c2 cells. Taken together, we demonstrate the sensitive detection of ROS by CL imaging in cultured cardiomyocytes.
Highlights
Reactive oxygen species (ROS) have been implicated in the pathogenesis of various human diseases, particular cardiac disorders [1, 2]
No CL responses were elicited by adding luminol/horseradish peroxidase (HRP) to the plate wells containing phosphate buffered saline (PBS) alone
ROS have been extensively implicated in the pathogenesis of cardiac disorders, studies on direct in situ CL
Summary
Reactive oxygen species (ROS) have been implicated in the pathogenesis of various human diseases, particular cardiac disorders [1, 2]. Because of the multiple cellular component nature of mammalian tissues, detection of cell-specific formation of ROS is of critical importance for understanding the oxidative mechanisms of disease process and for developing antioxidative stress-based strategies for intervention of oxidative tissue degeneration. In this context, over the last several decades various methods have been developed for detecting ROS formation in biological systems. Over the last several decades various methods have been developed for detecting ROS formation in biological systems These include the electron paramagnetic resonance (EPR) spin trapping, luminescent methods, as well as a number of biochemical assays [1, 5, 6]. In this study, we have applied the highly sensitive chemiluminescence (CL) imaging technique to investigate the in situ ROS formation in cultured monolayers of rat H9c2 cardiomyocytes and the factors that modulate the ROS generation in this widely used in vitro model for cardiac cell physiology and pathophysiology
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