Small-Molecule Inhibitors of Cyclophilins Block Opening of the Mitochondrial Permeability Transition Pore and Protect Mice From Hepatic Ischemia/Reperfusion Injury

Gastroenterology. 2019 Nov;157(5):1368-1382. doi: 10.1053/j.gastro.2019.07.026. Epub 2019 Jul 20.

Abstract

Background & aims: Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury.

Methods: We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif-/- (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis.

Results: The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2-16.2 μmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4-132 μmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif-/- mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle.

Conclusions: Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening.

Keywords: Drug; Mitochondrial Swelling; Mouse Model; PPIase Activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium Signaling / drug effects
  • Cells, Cultured
  • Cyclophilin D / antagonists & inhibitors*
  • Cyclophilin D / genetics
  • Cyclophilin D / metabolism
  • Cytoprotection
  • Disease Models, Animal
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Liver / drug effects*
  • Liver / enzymology
  • Liver / pathology
  • Liver Diseases / enzymology
  • Liver Diseases / genetics
  • Liver Diseases / pathology
  • Liver Diseases / prevention & control*
  • Male
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Mitochondria, Liver / drug effects*
  • Mitochondria, Liver / enzymology
  • Mitochondria, Liver / pathology
  • Mitochondrial Membrane Transport Proteins / antagonists & inhibitors*
  • Mitochondrial Membrane Transport Proteins / metabolism
  • Mitochondrial Permeability Transition Pore
  • Mitochondrial Swelling / drug effects
  • Reperfusion Injury / enzymology
  • Reperfusion Injury / genetics
  • Reperfusion Injury / pathology
  • Reperfusion Injury / prevention & control*
  • Signal Transduction

Substances

  • Cyclophilin D
  • Enzyme Inhibitors
  • Mitochondrial Membrane Transport Proteins
  • Mitochondrial Permeability Transition Pore
  • PPIF protein, mouse