In situ real-time chemiluminescence imaging of reactive oxygen species formation from cardiomyocytes

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.

Figure 1

Diagram depicting the highly sensitive chemiluminescence imaging system.

Figure 2

In situ real-time CL imaging of basal and BPQ-stimulated ROS formation in cultured monolayers of H9c2 cardiomyocytes. H9c2 cells were cultured in 6-well plates. Immediately prior to CL imaging, confluent cells in culture were washed once with PBS followed by addition of 2 mL PBS containing 1 μM BPQ or other reagents, as described under Materials and Methods section. (a) Representative CL images acquired at the indicated time points; the images for the first 10 minutes were acquired with a 5-minute delay; (b) layout of treatment groups; probe refers to luminol/HRP; (c) quantification of time-dependent ROS formation by luminol/HRP-amplified CL imaging. Data in (c) represent averages of two measurements.

Figure 3

Effects of exogenously added SOD/CAT on basal and BPQ-stimulated ROS formation in cultured monolayers of H9c2 cardiomyocytes. The experimental condition was the same as that described in the legend of Figure 2 except that SOD (250 units/mL) and CAT (250 units/mL) were added to the top 3 wells. (c) Shows the quantitative data of the images in (a).

Figure 4

In situ real-time CL imaging of ROS formation in cultured H9c2 cardiomyocytes exposed to inhibitors of mitochondrial ETC. H9c2 cells were cultured in 6-well plates. Immediately prior to CL imaging, confluent cells in culture were washed once with PBS followed by addition of 2 mL PBS containing 10 μM AA or ROT in the presence of probe (luminol/HRP), as described under Materials and Methods section. (a) Representative CL images acquired at the indicated time points; (b) layout of treatment groups, probe refers to luminol/HRP, (c) quantification of time-dependent ROS formation by luminol/HRP-amplified CL imaging. (c) Shows the quantitative data of the images in (a).

Figure 5

Induction of endogenous antioxidants by D3T in cultured H9c2 cardiomyocytes and schematic illustration of ROS detoxification by antioxidants. (a) H9c2 cells were incubated with 100 μM D3T in culture medium for 48 hours followed by measurement of the indicated antioxidants, as described under Materials and Methods section. Data represent mean ±SEM (n = 3-4).∗: significantly different from control; (b) detoxification of superoxide and H₂O₂ by various cellular antioxidants.

Figure 6

In situ real-time imaging of the effects of D3T pretreatment on basal and BPQ-stimulated ROS formation in cultured monolayers of H9c2 cardiomyocytes. H9c2 cells were treated with or without 100 μM D3T for 48 hours in culture medium before CL imaging experiment. For CL imaging, confluent cells in culture were washed once with PBS followed by addition of 2 mL PBS containing 0.2 and 1 μM BPQ or other reagents, as described under Materials and Methods section. (a) Representative CL images acquired at the indicated time points; (b) layout of treatment groups, probe refers to luminol/HRP, (c) quantification of time-dependent ROS formation by luminol/HRP-amplified CL imaging. Data in (c) represent averages of two measurements.