# In Situ Real-Time Chemiluminescence Imaging of  Reactive Oxygen Species Formation from Cardiomyocytes

Authors: Yunbo Li; Haiou Shen; Hong Zhu; Michael A. Trush; Ming Jiang; Ge Wang
Journal: International Journal of Biomedical Imaging
 (2008)

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## 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.


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## Body

## 1. Introduction

Reactive oxygen species (ROS) have
been implicated in the pathogenesis of various human diseases, particular
cardiac disorders [1, 2]. ROS include a number of oxygen-containing reactive
species, among which superoxide and hydrogen peroxide (H2O2)
are most widely studied in biomedical field [3, 4]. ROS attack cellular
constitutes, including lipids, proteins, and nucleic acids, leading to oxidative
stress and tissue injury. Due to the detrimental effects of ROS, mammalian
cells have evolved a number of antioxidants to protect cells from oxidative
injury. However, under pathophysiological conditions, ROS formation may
overwhelm the normal cellular antioxidant defenses, and as such oxidative
tissue injury and disease process occur [3, 4].There are a number of cellular
sources of ROS formation, which include mitochondrial electron transport chain
(ETC), NAD(P)H oxidase, uncoupled nitric oxide synthases, and cytochrome P450
enzymes [3, 4]. The above cellular sources of ROS formation are implicated in
oxidative stress of various tissues, including vasculature and myocardium
[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. These
include the electron paramagnetic resonance (EPR) spin trapping, luminescent
methods, as well as a number of biochemical assays [1, 5, 6]. While EPR spin
trapping technique is of the highest specificity for detecting oxygen radicals,
its sensitivity is limited [5, 6]. Moreover, EPR technique and most of the
biochemical assays for detecting ROS are unable to directly visualize the ROS formation
in a highly sensitive real-time manner. Direct sensitive in situ detection of
ROS formation from cultured cells provides important insight into the molecular
and biochemical mechanisms by which ROS mediate pathophysiological processes.
Accordingly, 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.


## 2. Materials and Methods

### 2.1. Chemical and Reagents

D3T with a purity of 99.8% was generously provided by Dr. Mary Tanga at
SRI International (Menlo Park, Calif, USA) and Dr. Linda Brady at the National
Institute of Mental Health (Bethesda, Md, USA).
Benzo(a)pyrene-1,6-quinone (BPQ) was from Midwest Research Institute (Kansas
City, Mo, USA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin,
streptomycin, and fetal bovine serum (FBS) were from Gibco-Invitrogen
(Carlsbad, Calif, USA). All other chemicals and reagents were from
Sigma-Aldrich (St. Louis, Mo, USA).


### 2.2. Cell Culture and Treatment

Rat H9c2 cardiomyocytes (ATCC,
Manassas, Va, USA) were cultured in DMEM supplemented with 10% FBS, 100 U/mL of
penicillin, and 100μg/mL of streptomycin in tissue culture
flasks at 37C° in a humidified atmosphere of 5%  CO2. The
cells were fed every 2-3 days, and subcultured once they reached 70–80% confluence.
For CL imaging experiments, 2×105cells were plated in each well of
6-well plates. The CL images were acquired directly with the confluent cells
(2.8×105cells/well or 0.3×105/cm2) in culture
at 37C° after washing of the cells with phosphate buffered saline
(PBS). To initiate the CL response, 10 μM luminol and 5 μg/mL
horseradish peroxidase (HRP) were added to the cell monolayers in 6-well
plates, with each well containing 2 mL PBS. For experiments on induction of
antioxidants, H9c2 cells were incubated with 100 μM D3T dissolved in dimethyl sulfoxide
(DMSO; 0.1% final concentration) in culture medium for 48 hours. Control cells
received 0.1% DMSO only.


### 2.3. Cell Extract Preparation

H9c2 cells were collected and
resuspended in ice-cold 50 mM potassium phosphate buffer, pH 7.4,
containing 2 mM EDTA and 0.1% Triton X-100. The cells were sonicated, followed
by centrifugation at 13 000 g for 10 minutes at4C°. The
resulting supernatants were collected, and the protein concentrations were
quantified with Bio-Rad protein assay dye (Hercules, Calif, USA) using bovine
serum albumin as the standard. The samples were kept on ice for measurement of
the antioxidants as described below.


### 2.4. Measurement of Cellular Antioxidants

Cellular superoxide dismutase (SOD)
activity was determined by the method of Spitz and Oberley, as described before
[7, 8]. The cellular SOD activity was calculated using a concurrently run
Cu,ZnSOD (Sigma-Aldrich) standard curve, and expressed as units per mg of cellular protein.Cellular NAD(P)H:quinone
oxidoreductase 1 (NQO1) activity was determined using dichloroindophenol (DCIP)
as the two-electron acceptor and NADPH as the electron donor in the presence or
absence of dicumarol, as described before [7]. The dicumarol-sensitive NQO1
activity was calculated using the extinction coefficient of 21.0mM−1cm−1,
and expressed as nmol of DCIP reduced per minute per mg of cellular
protein.Cellular catalase activity was determined by measuring the
decomposition ofH2O2 at 240 nm, according to the method
of Aebi [9], and expressed as μmol of H2O2 consumed per minute per mg of cellular protein.Cellular GSH content was determined
by measuring the formation of the fluorescent conjugate from reaction of GSH
witho-phthalaldehyde according to
the procedure described previously [7]. Cellular GSH content was calculated
using a concurrently run GSH (Sigma-Aldrich) standard curve, and expressed as
nmol of GSH per mg of cellular protein.Cellular glutathione reductase (GR)
activity was measured according to the method initially reported by Wheeler et
al. [10] with modifications, as described previously [9]. GR activity was
calculated using the extinction coefficient of 6.22mM−1cm−1,
and expressed as nmol of NADPH consumed per minute per mg of cellular
protein.Cellular glutathione peroxidase
(GPx) activity was determined by the method of Flohé and Günzler [11] with
slight modifications, as described before [12]. This assay is based on the
formation of GSSG from GPx-catalyzed oxidation of GSH by H2O2,
coupled with NADPH consumption in the presence of exogenously added GR. GPx
activity was calculated using the extinction coefficient of 6.22mM−1cm−1,
and expressed as nmol of NADPH consumed per minute per mg of cellular protein.


### 2.5. in Situ Real-Time Cl Imaging

As shown in Figure1, a liquid
nitrogen cooled back-illuminated charge-coupled device (CCD) camera (VersArray
2048B, Princeton Instruments, Trenton, NJ, USA) with a Nikon 50 mm f/1.2 lens
was used to image the 6-well cell culture plates. The CCD sensor has a
27.6×27.6mm imaging area with 2048×2048pixels. It has a >80% quantum
efficiency for visible spectrum range. To capture the ultra low light image, a
hardware binning of 8×8 was selected to increase the sensitivity by 64 times.
To reduce the noise, the CCD sensor was cooled to −110C°, and a 50 kHz read out
frequency was used to minimize the dark current and read out noise. The culture
plates were placed on a heated plate (37C°). An enhanced aluminum front mirror
was placed above the culture plates to redirect the photons to the camera. To
ensure that the imaging experiment environment is totally dark, the imaging
system is placed in a light tight enclosure. After placing the culture
plates in the imaging system, we immediately took 6 CL images, each with ten
minutes of exposure time.Figure 1
Diagram depicting the highly sensitive chemiluminescence imaging system.Before we extracted out the data, we
calibrated the intensity of the CCD sensor by using dark frames to remove the
background and thermal noise in a totally dark environment. Each dark frame was
collected with the same integral time at the same cooling temperature in the
experiment. A master dark frame was then computed by averaging 20 dark frames.
For each CL image, we subtracted the master dark frame from it to get the final
image. For each well in the culture plates, we summed the pixel value inside
the well and divided it by the number of pixels in the well to get an average
intensity for each well.


### 2.6. Statistical Analysis

Data
are expressed as means±SEM from at least 3 separate experiments unless
indicated otherwise. Differences between 2 groups were analyzed by Student’s t-test. Statistical significance was considered at P<.05.


## 2.1. Chemical and Reagents

D3T with a purity of 99.8% was generously provided by Dr. Mary Tanga at
SRI International (Menlo Park, Calif, USA) and Dr. Linda Brady at the National
Institute of Mental Health (Bethesda, Md, USA).
Benzo(a)pyrene-1,6-quinone (BPQ) was from Midwest Research Institute (Kansas
City, Mo, USA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin,
streptomycin, and fetal bovine serum (FBS) were from Gibco-Invitrogen
(Carlsbad, Calif, USA). All other chemicals and reagents were from
Sigma-Aldrich (St. Louis, Mo, USA).


## 2.2. Cell Culture and Treatment

Rat H9c2 cardiomyocytes (ATCC,
Manassas, Va, USA) were cultured in DMEM supplemented with 10% FBS, 100 U/mL of
penicillin, and 100μg/mL of streptomycin in tissue culture
flasks at 37C° in a humidified atmosphere of 5%  CO2. The
cells were fed every 2-3 days, and subcultured once they reached 70–80% confluence.
For CL imaging experiments, 2×105cells were plated in each well of
6-well plates. The CL images were acquired directly with the confluent cells
(2.8×105cells/well or 0.3×105/cm2) in culture
at 37C° after washing of the cells with phosphate buffered saline
(PBS). To initiate the CL response, 10 μM luminol and 5 μg/mL
horseradish peroxidase (HRP) were added to the cell monolayers in 6-well
plates, with each well containing 2 mL PBS. For experiments on induction of
antioxidants, H9c2 cells were incubated with 100 μM D3T dissolved in dimethyl sulfoxide
(DMSO; 0.1% final concentration) in culture medium for 48 hours. Control cells
received 0.1% DMSO only.


## 2.3. Cell Extract Preparation

H9c2 cells were collected and
resuspended in ice-cold 50 mM potassium phosphate buffer, pH 7.4,
containing 2 mM EDTA and 0.1% Triton X-100. The cells were sonicated, followed
by centrifugation at 13 000 g for 10 minutes at4C°. The
resulting supernatants were collected, and the protein concentrations were
quantified with Bio-Rad protein assay dye (Hercules, Calif, USA) using bovine
serum albumin as the standard. The samples were kept on ice for measurement of
the antioxidants as described below.


## 2.4. Measurement of Cellular Antioxidants

Cellular superoxide dismutase (SOD)
activity was determined by the method of Spitz and Oberley, as described before
[7, 8]. The cellular SOD activity was calculated using a concurrently run
Cu,ZnSOD (Sigma-Aldrich) standard curve, and expressed as units per mg of cellular protein.Cellular NAD(P)H:quinone
oxidoreductase 1 (NQO1) activity was determined using dichloroindophenol (DCIP)
as the two-electron acceptor and NADPH as the electron donor in the presence or
absence of dicumarol, as described before [7]. The dicumarol-sensitive NQO1
activity was calculated using the extinction coefficient of 21.0mM−1cm−1,
and expressed as nmol of DCIP reduced per minute per mg of cellular
protein.Cellular catalase activity was determined by measuring the
decomposition ofH2O2 at 240 nm, according to the method
of Aebi [9], and expressed as μmol of H2O2 consumed per minute per mg of cellular protein.Cellular GSH content was determined
by measuring the formation of the fluorescent conjugate from reaction of GSH
witho-phthalaldehyde according to
the procedure described previously [7]. Cellular GSH content was calculated
using a concurrently run GSH (Sigma-Aldrich) standard curve, and expressed as
nmol of GSH per mg of cellular protein.Cellular glutathione reductase (GR)
activity was measured according to the method initially reported by Wheeler et
al. [10] with modifications, as described previously [9]. GR activity was
calculated using the extinction coefficient of 6.22mM−1cm−1,
and expressed as nmol of NADPH consumed per minute per mg of cellular
protein.Cellular glutathione peroxidase
(GPx) activity was determined by the method of Flohé and Günzler [11] with
slight modifications, as described before [12]. This assay is based on the
formation of GSSG from GPx-catalyzed oxidation of GSH by H2O2,
coupled with NADPH consumption in the presence of exogenously added GR. GPx
activity was calculated using the extinction coefficient of 6.22mM−1cm−1,
and expressed as nmol of NADPH consumed per minute per mg of cellular protein.


## 2.5. in Situ Real-Time Cl Imaging

As shown in Figure1, a liquid
nitrogen cooled back-illuminated charge-coupled device (CCD) camera (VersArray
2048B, Princeton Instruments, Trenton, NJ, USA) with a Nikon 50 mm f/1.2 lens
was used to image the 6-well cell culture plates. The CCD sensor has a
27.6×27.6mm imaging area with 2048×2048pixels. It has a >80% quantum
efficiency for visible spectrum range. To capture the ultra low light image, a
hardware binning of 8×8 was selected to increase the sensitivity by 64 times.
To reduce the noise, the CCD sensor was cooled to −110C°, and a 50 kHz read out
frequency was used to minimize the dark current and read out noise. The culture
plates were placed on a heated plate (37C°). An enhanced aluminum front mirror
was placed above the culture plates to redirect the photons to the camera. To
ensure that the imaging experiment environment is totally dark, the imaging
system is placed in a light tight enclosure. After placing the culture
plates in the imaging system, we immediately took 6 CL images, each with ten
minutes of exposure time.Figure 1
Diagram depicting the highly sensitive chemiluminescence imaging system.Before we extracted out the data, we
calibrated the intensity of the CCD sensor by using dark frames to remove the
background and thermal noise in a totally dark environment. Each dark frame was
collected with the same integral time at the same cooling temperature in the
experiment. A master dark frame was then computed by averaging 20 dark frames.
For each CL image, we subtracted the master dark frame from it to get the final
image. For each well in the culture plates, we summed the pixel value inside
the well and divided it by the number of pixels in the well to get an average
intensity for each well.


## 2.6. Statistical Analysis

Data
are expressed as means±SEM from at least 3 separate experiments unless
indicated otherwise. Differences between 2 groups were analyzed by Student’s t-test. Statistical significance was considered at P<.05.


## 3. RESULTS

### 3.1. Detection of Basal and Bpq-Stimulated Ros Formation by Cl Imaging in Cultured Monolayers of H9c2 Cells

As shown in Figure2, incubation of
cultured monolayers of H9c2 cells with luminol/HRP led to CL responses as
detected by the highly sensitive imaging system (see Figure 1), indicating that
H9c2 cells in culture could constitutively release ROS. Notably, no CL
responses were elicited by adding luminol/HRP to the plate wells containing PBS
alone (data not shown). In addition, under our experimental conditions,
viability of H9c2 cells in cultures was >99% based on trypan blue exclusion
assay (data not shown). Dramatically augmented CL responses were observed after
addition of 1 μM BPQ, a mitochondrial redox cycler, to
the cells in culture, and the CL responses remained elevated for up to 1 hour.
The intensity of the BPQ-stimulated CL responses was 4-5 times that of basal CL
responses (see Figure 2). Both constitutive and BPQ-induced CL responses were
completely abolished in the presence of exogenously added SOD (250 units/mL)
and catalase (250 units/mL) (see Figure 3).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 1μM
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 Figure2 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).


### 3.2. Detection by Cl Imaging of The Effects of Mitochondrial Etc Inhibitors on Ros Formation in Cultured Monolayers of H9c2 Cells

To
further investigate the involvement of mitochondria in cellular ROS release,
H9c2 cells in culture were exposed to two commonly used mitochondrial
inhibitors, antimycin A (AA) and rotenone (ROT). As shown in Figure4, AA
treatment slightly decreased the CL responses, whereas exposure to ROT led to
small increases in the CL responses. The marginal effects by either AA or ROT
on the CL responses were only obvious during the first 30 minutes of treatment.
From 30 to 60 minutes, the CL responses among the 3 treatment groups were
essentially the same (see Figure 4). AA and ROT were dissolved in DMSO (0.1%).
Control cells received 0.1% DMSO only.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 10μM
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).


### 3.3. Induction of Cellular Antioxidants by D3t and The Effects on Basal and Bpq-Stimulated Ros Formation in Cultured Monolayers of H9c2 Cells

As
shown in Figure5, incubation of H9c2 cells with 100 μM
D3T for 48 hours resulted in significant induction of cellular NQO1, CAT, GSH,
GR, and GPx. These antioxidants are crucially involved in detoxification of
ROS, including superoxide and H2O2 [3]. To determine if
upregulation of cellular antioxidants by D3T could blunt ROS formation by H9c2
cells, the cardiomyocytes in culture were first treated with D3T and then
exposed to BPQ. As shown in Figure 6, D3T pretreatment resulted in marked
inhibition of BPQ-stimulated ROS formation. Notably, D3T pretreatment also
dramatically decreased the constitutive formation of ROS by cultured H9c2
cells. The inhibitory effects of D3T pretreatment on both constitutive and
BPQ-stimulated ROS formation were sustained during the 1-hour period of
experiment (see Figure 6). Also as shown in Figure 6, in contrast to 1 μM
BPQ, 0.2 μM BPQ-elicited CL responses decreased
over 60 minutes in untreated cell cultures.Induction of endogenous antioxidants by D3T in cultured H9c2 cardiomyocytes and
schematic illustration of ROS detoxification by antioxidants. (a) H9c2 cells
were incubated with 100μM 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.
(a)(b)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 100μM 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.


## 3.1. Detection of Basal and Bpq-Stimulated Ros Formation by Cl Imaging in Cultured Monolayers of H9c2 Cells

As shown in Figure2, incubation of
cultured monolayers of H9c2 cells with luminol/HRP led to CL responses as
detected by the highly sensitive imaging system (see Figure 1), indicating that
H9c2 cells in culture could constitutively release ROS. Notably, no CL
responses were elicited by adding luminol/HRP to the plate wells containing PBS
alone (data not shown). In addition, under our experimental conditions,
viability of H9c2 cells in cultures was >99% based on trypan blue exclusion
assay (data not shown). Dramatically augmented CL responses were observed after
addition of 1 μM BPQ, a mitochondrial redox cycler, to
the cells in culture, and the CL responses remained elevated for up to 1 hour.
The intensity of the BPQ-stimulated CL responses was 4-5 times that of basal CL
responses (see Figure 2). Both constitutive and BPQ-induced CL responses were
completely abolished in the presence of exogenously added SOD (250 units/mL)
and catalase (250 units/mL) (see Figure 3).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 1μM
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 Figure2 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).


## 3.2. Detection by Cl Imaging of The Effects of Mitochondrial Etc Inhibitors on Ros Formation in Cultured Monolayers of H9c2 Cells

To
further investigate the involvement of mitochondria in cellular ROS release,
H9c2 cells in culture were exposed to two commonly used mitochondrial
inhibitors, antimycin A (AA) and rotenone (ROT). As shown in Figure4, AA
treatment slightly decreased the CL responses, whereas exposure to ROT led to
small increases in the CL responses. The marginal effects by either AA or ROT
on the CL responses were only obvious during the first 30 minutes of treatment.
From 30 to 60 minutes, the CL responses among the 3 treatment groups were
essentially the same (see Figure 4). AA and ROT were dissolved in DMSO (0.1%).
Control cells received 0.1% DMSO only.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 10μM
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).


## 3.3. Induction of Cellular Antioxidants by D3t and The Effects on Basal and Bpq-Stimulated Ros Formation in Cultured Monolayers of H9c2 Cells

As
shown in Figure5, incubation of H9c2 cells with 100 μM
D3T for 48 hours resulted in significant induction of cellular NQO1, CAT, GSH,
GR, and GPx. These antioxidants are crucially involved in detoxification of
ROS, including superoxide and H2O2 [3]. To determine if
upregulation of cellular antioxidants by D3T could blunt ROS formation by H9c2
cells, the cardiomyocytes in culture were first treated with D3T and then
exposed to BPQ. As shown in Figure 6, D3T pretreatment resulted in marked
inhibition of BPQ-stimulated ROS formation. Notably, D3T pretreatment also
dramatically decreased the constitutive formation of ROS by cultured H9c2
cells. The inhibitory effects of D3T pretreatment on both constitutive and
BPQ-stimulated ROS formation were sustained during the 1-hour period of
experiment (see Figure 6). Also as shown in Figure 6, in contrast to 1 μM
BPQ, 0.2 μM BPQ-elicited CL responses decreased
over 60 minutes in untreated cell cultures.Induction of endogenous antioxidants by D3T in cultured H9c2 cardiomyocytes and
schematic illustration of ROS detoxification by antioxidants. (a) H9c2 cells
were incubated with 100μM 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.
(a)(b)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 100μM 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.


## 4. DISCUSSION

Although
ROS have been extensively implicated in the pathogenesis of cardiac disorders,
studies on direct in situ CL imaging of ROS formation in cultured cardiac cells
are lacking in the literature. In this study, we have applied a highly
sensitive CL imaging system (see Figure1) to investigate the in situ real-time
ROS formation in cultured monolayers of rat H9c2 cardiomyocytes, a widely used
in vitro cell model for studying cardiac cell biology and cytoprotection
[13, 14]. By using this innovative CL imaging system, we have demonstrated that
significant amounts of ROS could be released from H9c2 cells in culture under
unstimulated conditions (see Figure 2). While the exact cellular sources for
this constitutive ROS formation in H9c2 cells remain to be elucidated, one
possible site could be the mitochondrial ETC. In this regard, mitochondria account
for roughly 40–50% of the total
mass of cardiomyocytes. Considering the high mitochondrial activity in
cardiomyocytes, we next determined if BPQ could stimulate the ROS formation
from H9c2 cells in culture. Indeed, dramatically augmented CL responses were observed after adding 1 μM, and even 0.2 μM BPQ to the cultured monolayers of H9c2 cells (see Figures 2
and 6), suggesting that BPQ is a potent ROS generator in cardiomyocytes. It
remains unclear why the CL responses elicited by 0.2 μM
BPQ decreased over 60 minutes in untreated cell cultures (see Figure 6). It
might be due to the significant detoxification of the small amount of BPQ (0.4nmol/2.8×105cells) in H9c2 cells, leading to decreased availability of the free BPQ
molecules that undergo redox cycling to produce ROS.BPQ is a quinone metabolite derived
from benzo(a)pyrene, an environmental pollutant implicated in cardiovascular
diseases [15, 16]. Benzo(a)pyrene is also present in cigarette smoking, which is
a major risk factor for human cardiovascular diseases [15, 17]. Metabolism of
benzo(a)pyrene by mammalian tissues, including cardiovascular tissue, can form
significant amounts of BPQ [18]. We have previously demonstrated that BPQ
preferentially undergoes redox cycling in mitochondria, leading to ROS
formation [6, unpublished observation].
Thus, the mitochondrial ETC could also be the site where BPQ underwent
redox cycling to generate ROS in the cultured monolayers of H9c2 cells. To
further investigate the involvement of mitochondrial ETC in ROS formation by
H9c2 cells, AA and ROT were used to selectively block the electron transport at
complexes I and III, respectively [19]. The marginal effects of either AA or
ROS on ROS formation by H9c2 cells (see Figure 4) suggested that blocking the mitochondrial
ETC was not an effective mechanism for altering ROS formation by H9c2 cells.
The redox cycling of BPQ thus appears to be a much more effective pathway for
augmentation of ROS formation by cardiomyocytes. This is an important
observation considering the involvement of benzo(a)pyrene in cardiovascular
diseases. The potent and sustained stimulation of ROS formation by BPQ in
cardiomyocytes would create significant oxidative stress in myocardium. In this
context, augmented ROS formation and the subsequent oxidative cell injury in
myocardium are crucial events underlying cardiac disorders [1, 2]. Likewise,
inhibition or scavenging of ROS in myocardium has been shown to be an effective
strategy for intervention of oxidative cardiac injury [20, 21].In this study, we also
demonstrated that pretreatment with D3T significantly upregulated cellular antioxidants
and markedly inhibited both constitutive and BPQ-stimulated ROS formation in
cultured monolayers of H9c2 cells (see Figure6). D3T is a cruciferous
sulfur-containing compound, which potently induces antioxidants in mammalian
cells/tissues [22]. In H9c2 cells, D3T potently upregulated NQO1, CAT, GSH, GR,
and GPX (see Figure 5). NOQ1 is recently found to scavenge superoxide [23].
CAT, GSH, GR, and GPx are key cellular factors primarily for detoxification of H2O2 [3]. As illustrated in Figure 5(b), coordinated
induction of the above cellular antioxidants in H9c2 cells would enhance
detoxification of both superoxide and H2O2, and thereby
leading to the decreases in luminol/HRP-amplified CL responses. This is the
first study demonstrating that upregulation of cellular antioxidants by D3T was
accompanied by markedly decreased oxidative stress induced by BPQ in cultured
cardiomyocytes. The above finding also implicated D3T as a promising
chemoprotective nutraceutical for intervention of oxidative cardiac injury
associated with exposure to benzo(a)pyrene as well as other pollutants that
give rise to redox-cycling
quinones. In this regard, consumption of cruciferous vegetables rich in
organosulfur compounds, including dithiolethiones, is associated with decreased incidence of
cardiovascular events [24, 25].In summary, by using the highly
sensitive CL imaging system, we demonstrate that cultured monolayers of H9c2
cardiomyocytes constitutively release ROS under physiological conditions, and
the redox cycler
BPQ potently stimulates the ROS formation in the cultured cardiomyocytes. With
this innovative imaging system, we also demonstrate that upregulation of
cellular antioxidants by cruciferous D3T appears to be a highly effective
strategy for mitigating oxidative stress induced by BPQ in the cultured
cardiomyocytes. These observations lay a basis for investigation of myocardial
oxidative stress as well as cardioprotection in animal models. Although most of
the data presented in this work were from 2 independent measurements, our
ongoing research on luminol/HRP-based CL imaging further confirms the ability
of this innovative technique to
reproducibly detect ROS in both cellular systems and in vivo animals.



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*Source: 941729-2009-02-25.xml*