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Published on: October 27, 2020
Moesin functionality in hypothermic liver preservation injury
Tao Tian1, Susanne L Lindell1, Chris Kowalski1
1Department of Surgery, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA 23298, United States.
Abstract:
The objective of this study was to determine how expression and functionality of the cytoskeletal linker protein moesin is involved in hepatic hypothermic preservation injury. Mouse livers were cold stored in University of Wisconsin (UW) solution and reperfused on an isolated perfused liver (IPL) device for one hour. Human hepatocytes (HepG2) and human or murine sinusoidal endothelial cells (SECs) were cold stored and rewarmed to induce hypothermic preservation injury. The cells were transfected with: wild type moesin, an siRNA duplex specific for moesin, and the moesin mutants T558D and T558A. Tissue and cell moesin expression and its binding to actin were determined by Western blot. Liver IPL functional outcomes deteriorated proportional to the length of cold storage, which correlated with moesin disassociation from the actin cytoskeleton. Cell viability (LDH and WST-8) in the cell models progressively declined with increasing preservation time, which also correlated with moesin disassociation. Transfection of a moesin containing plasmid or an siRNA duplex specific for moesin into HepG2 cells resulted in increased and decreased moesin expression, respectively. Overexpression of moesin protected while moesin knock-down potentiated preservation injury in the HepG2 cell model. Hepatocytes expressing the T558A (inactive) and T558D (active) moesin binding mutants demonstrated significantly more and less preservation injury, respectively. Cold storage time dependently caused hepatocyte detachment from the matrix and cell death, which was prevented by the T558D active moesin mutation. In conclusion, moesin is causally involved in hypothermic liver cell preservation injury through control of its active binding molecular functionality.
Insights
Moesin protein is crucial for liver preservation. Its active form protects liver cells from damage during cold storage, while its inactivation worsens injury, highlighting its role in preventing cell death.
Area of Science:
- Hepatology
- Cell Biology
- Biochemistry
Background:
- Hypothermic preservation is essential for organ transplantation but can cause liver injury.
- The role of cytoskeletal proteins, like moesin, in this injury is not fully understood.
Purpose of the Study:
- To investigate the involvement of moesin expression and function in hepatic hypothermic preservation injury.
- To elucidate moesin's role in maintaining liver cell integrity during cold storage.
Main Methods:
- Mouse livers and human/murine cells (hepatocytes, sinusoidal endothelial cells) underwent cold storage and reperfusion/rewarming.
- Moesin expression, actin binding, and cell viability were assessed after transfection with wild-type moesin, moesin siRNA, or moesin mutants (T558D, T558A).
- Western blot, LDH, and WST-8 assays were employed to evaluate moesin levels, actin association, and cell injury.
Main Results:
- Liver function and cell viability declined with increased cold storage time, correlating with moesin dissociation from actin.
- Moesin overexpression protected cells from preservation injury, whereas moesin knockdown exacerbated it.
- The active moesin mutant (T558D) prevented hepatocyte detachment and cell death, unlike the inactive mutant (T558A).
Conclusions:
- Moesin plays a causal role in hypothermic liver cell preservation injury.
- Moesin's active binding functionality is key to protecting hepatocytes from cold storage-induced damage.
- Targeting moesin activity may offer a therapeutic strategy to improve organ preservation outcomes.
