Mitochondrial Dysfunction and Autophagy in Hepatic Ischemia/Reperfusion Injury

Abstract

Ischemia/reperfusion (I/R) injury remains a major complication of liver resection, transplantation, and hemorrhagic shock. Although the mechanisms that contribute to hepatic I/R are complex and diverse involving the interaction of cell injury in hepatocytes, immune cells, and endothelium, mitochondrial dysfunction is a cardinal event culminating in hepatic reperfusion injury. Mitochondrial autophagy, so-called mitophagy, is a key cellular process that regulates mitochondrial homeostasis and eliminates damaged mitochondria in a timely manner. Growing evidence accumulates that I/R injury is attributed to defective mitophagy. This review aims to summarize the current understanding of autophagy and its role in hepatic I/R injury and highlight the various therapeutic approaches that have been studied to ameliorate injury.

Figure 1

Onset of the MPT after I/R in primary rodent hepatocytes. After 4 hours of simulated ischemia, hepatocytes were reperfused and confocal images of calcein, tetramethylrhodamine methyl ester (TMRM), and propidium iodide (PI, arrows) were simultaneously collected. Polarized mitochondria take up red fluorescing TMRM while simultaneously excluding green fluorescing calcein due to the closed conformation of permeability transition pores. After 4 hours of ischemia, anoxia depolarized the mitochondria and TMRM fluorescence was undetectable. At the same time, the mitochondria in the green channel appeared as dark and round voids where each void represents a single, polarized mitochondrion, indicative of the absence of MPT onset during ischemia. After reperfusion, the mitochondria transiently repolarized within 10 minutes, but the MPT initiated thereafter, as shown by the loss of TMRM fluorescence and diffusion of cytosolic calcein into the mitochondria. Both calcein and TMRM fluorescence completely vanished at 50 minutes and PI labeled the nuclei (arrows) due to the loss of the plasma membrane integrity.

Figure 2

A schematic of autophagy process.

Figure 3

Autophagy in primary mouse hepatocytes. (a) Confocal microscopy with green fluorescent protein-labeled microtubule-associated protein 1 light chain 3 (GFP-LC3) and TMRM in normoxic hepatocytes. Under the basal condition of normoxia, GFP-LC3 predominantly localizes in the cytosol. After autophagy induction, hepatocytes show numerous punctate GFP-LC3, indicative of autophagosomes. Note that some red fluorescing mitochondria are entrapped by GFP-LC3, an event signifying the onset of mitophagy. The bottom panels represent magnified images of the square inserts at the top panels. (b) Loss of autophagy after I/R. Hepatocytes were labeled with GFP-LC3 and subjected to 4 hours of simulated ischemia. After 5 minutes of reperfusion, some autophagosomes (green fluorescing punctate structures) were evident but unable to sequester abnormal mitochondria (top panels). This cell was dead after 17 minutes, as indicated by PI labeling in the nuclei (yellow arrows). In striking contrast, when autophagy was stimulated prior to ischemia, hepatocytes executed a robust autophagy to clear abnormal mitochondria and remained viable after 2 hours of reperfusion. Empty arrows display the autophagosomes surrounding the mitochondria.

Figure 4

Scheme of I/R-induced impairment of autophagy. After reperfusion, hepatocytes become overloaded with ROS and calcium, which in turn stimulates calpains. These enzymes subsequently hydrolyze ATG7 and BECN1, causing defective autophagy. Since impaired autophagy fails to eliminate abnormal mitochondria, the mitochondria laden with ROS and calcium undergo the MPT and ultimately induce cell death. Suppression of calcium increase, inhibition of calpains with acetyl-leu-leu-methioninal, enhancement of autophagy, or blockade of the MPT with cyclosporin A or nitric oxide prevents reperfusion-induced cell death. Damaged hepatocytes subsequently release damage-associated molecular patterns and ROS. Kupffer cells and sinusoidal endothelial cells are then activated, leading to chemokine and cytokine release, neutrophil and platelet activation, and platelet adhesion to the sinusoidal lumen. Congestion and constriction of the sinusoid further aggravate reperfusion injury. AdATG7, adenovirus expressing ATG7; AdBECN1, adenovirus expressing Beclin-1; ALLM, acetyl-leu-leu-methioninal; CsA, cyclosporin A; DAMP, damage-associated molecular patterns; HO-1, heme oxygenase-1; ICAM-1, intercellular adhesion molecule-1; Mac-1, macrophage-1 antigen; NO, nitric oxide; ROS, reactive oxygen species.