Liver mitochondrial calcium uptake following cold ischemia is regulated by phospholipase C

Abstract

Introduction: Altered mitochondrial Ca2+ (MCa2+) uptake has been implicated in graft dysfunction following cold ischemia (CI), when excessive cytosolic Ca2+ enters mitochondria through the MCa2+ uniporter. The actual mechanisms governing uniporter activity are unknown. Ca2+ uptake by the cell is regulated by Phospholipase C (PLC). We therefore hypothesized that PLC exists in mitochondria and regulates MCa2+ uptake following CI. Methods: Rat livers were perfused with UW solution and harvested. Half was homogenized immediately; the other half was first subjected to 24 hour cold storage in UW. Mitochondria were isolated and incubated in a buffer containing 1mM ATP (known [ATP] during ischemia) and 0.2 μM 45Ca2+. Ruthenium Red (RR, [10 μM]) was used as a negative control. A selective PLC inhibitor, U-73122, and its inactive analog, U-73343, were added to determine their effects on MCa2+ uptake. Western blots were performed using anti-PLC antibodies. Mitochondrial transmembrane potential (MΔψ) was evaluated using Mitotracker Red fluorescence. Results: 1) Western blot confirmed the presence of PLC in isolated mitochondria and mitochondrial membranes. 2) MCa2+ uptake was significantly increased after 24 hour cold storage in UW. 3) U-73122 and RR, but not U-73343, significantly and dose-dependently decreased Ca2+ uptake in mitochondria from both ischemic and non-ischemic livers, without affecting MΔψ. Conclusions: These data demonstrate for the first time that PLC exists in liver mitochondria. The effects of U-73122 indicate that PLC is essential for MCa2+ uptake during both physiologic and CI conditions. Inhibition of mitochondrial PLC therefore represents a novel target in the study of CI damage during liver transplantation.