Coronary Endothelium No-Reflow Injury Is Associated with ROS-Modified Mitochondrial Fission through the JNK-Drp1 Signaling Pathway

Abstract

Coronary artery no-reflow is a complex problem in the area of reperfusion therapy, and the molecular mechanisms underlying coronary artery no-reflow injury have not been fully elucidated. In the present study, we explored whether oxidative stress caused damage to coronary endothelial cells by inducing mitochondrial fission and activating the JNK pathway. The hypoxia/reoxygenation (H/R) model was induced in vitro to mimic coronary endothelial no-reflow injury, and mitochondrial fission, mitochondrial function, and endothelial cell viability were analyzed using western blotting, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence. Our data indicated that reactive oxygen species (ROS) were significantly induced upon H/R injury, and this was followed by decreased endothelial cell viability. Mitochondrial fission was induced and mitochondrial bioenergetics were impaired in cardiac endothelial cells after H/R injury. Neutralization of ROS reduced mitochondrial fission and protected mitochondrial function against H/R injury. Our results also demonstrated that ROS stimulated mitochondrial fission via JNK-mediated Drp1 phosphorylation. These findings indicate that the ROS-JNK-Drp1 signaling pathway may be one of the molecular mechanisms underlying endothelial cell damage during H/R injury. Novel treatments for coronary no-reflow injury may involve targeting mitochondrial fission and the JNK-Drp1 signaling pathway.

Figure 1

Hypoxia/reoxygenation injury mediates endothelial cell oxidative stress. (a) Cell viability was measured using the CCK-8 assay. Cardiac endothelial cells (CECs) underwent hypoxia/reoxygenation (H/R) injury. (b–e) The content of antioxidative factors was measured using an enzyme-linked immunosorbent assay (ELISA). (f, g) Intracellular ROS and mitochondrial ROS (mROS) were detected using DCFHDA and MitoSOX red mitochondrial superoxide indicator, respectively. ^∗p < 0.05.

Figure 2

Mitochondrial ROS promotes Drp1 phosphorylation and mitochondrial fission in endothelial cells under hypoxia/reoxygenation injury. (a, b) Mitochondrial fission was measured using immunofluorescence. Cardiac endothelial cells (CECs) underwent hypoxia/reoxygenation (H/R) injury. Mito-Q, a mitochondrial antioxidant, was added to the CEC media to neutralize mROS. (c–e) qPCR was performed to analyze the transcription of Drp1, Mff, and Fis1 in CECs under H/R injury. ^∗p < 0.05.

Figure 3

Mitochondrial dysfunction is induced by mitochondrial ROS. (a) Adenosine triphosphate (ATP) production was measured using an enzyme-linked immunosorbent assay (ELISA). Cardiac endothelial cells (CECs) underwent hypoxia/reoxygenation (H/R) injury. Mito-Q, a mitochondrial antioxidant, was added to the CEC media to neutralize mROS. (b–d) The activity of the mitochondrial respiration complex was determined using an ELISA. ^∗p < 0.05.

Figure 4

Inhibition of mitochondrial fission also sustains mitochondrial function in endothelial cells. (a) Adenosine triphosphate (ATP) production was measured using an enzyme-linked immunosorbent assay (ELISA). Cardiac endothelial cells (CECs) underwent hypoxia/reoxygenation (H/R) injury. Mdivi-1, a mitochondrial fission inhibitor, was added to the CEC culture media to repress mitochondrial fission. (b–d) The activity of the mitochondrial respiration complex was determined using an ELISA. ^∗p < 0.05.

Figure 5

ROS causes Drp1-related mitochondrial fission through the JNK pathway. (a–c) Proteins were isolated from hypoxia/reoxygenation- (H/R-) treated CECs. Western blotting was performed to analyze the expression of JNK and Drp1. Mito-Q, a mitochondrial antioxidant, was added to the CEC culture media to neutralize mROS. (d–f) Immunofluorescence was performed to verify the expressions of JNK and Drp1. Mito-Q, a mitochondrial antioxidant, was added to the CEC culture media to neutralize mROS. ^∗p < 0.05.