title: Carbamazepine suppresses calpain-mediated autophagy impairment after ischemia/reperfusion in mouse livers.
abstract: Onset of the mitochondrial permeability transition (MPT) plays a causative role in ischemia/reperfusion (I/R) injury. Current therapeutic strategies for reducing reperfusion injury remain disappointing. Autophagy is a lysosome-mediated, catabolic process that timely eliminates abnormal or damaged cellular constituents and organelles such as dysfunctional mitochondria. I/R induces calcium overloading and calpain activation, leading to degradation of key autophagy-related proteins (Atg). Carbamazepine (CBZ), an FDA-approved anticonvulsant drug, has recently been reported to increase autophagy. We investigated the effects of CBZ on hepatic I/R injury. Hepatocytes and livers from male C57BL/6 mice were subjected to simulated in vitro, as well as in vivo I/R, respectively. Cell death, intracellular calcium, calpain activity, changes in autophagy-related proteins (Atg), autophagic flux, MPT and mitochondrial membrane potential after I/R were analyzed in the presence and absence of 20 μM CBZ. CBZ significantly increased hepatocyte viability after reperfusion. Confocal microscopy revealed that CBZ prevented calcium overloading, the onset of the MPT and mitochondrial depolarization. Immunoblotting and fluorometric analysis showed that CBZ blocked calpain activation, depletion of Atg7 and Beclin-1 and loss of autophagic flux after reperfusion. Intravital multiphoton imaging of anesthetized mice demonstrated that CBZ substantially reversed autophagic defects and mitochondrial dysfunction after I/R in vivo. In conclusion, CBZ prevents calcium overloading and calpain activation, which, in turn, suppresses Atg7 and Beclin-1 depletion, defective autophagy, onset of the MPT and cell death after I/R.
authors.affiliation: Department of Surgery, University of Florida, Gainesville, FL 32610, USA. Electronic address: Jae.Kim@surgery.ufl.edu.
authors.forename: Jae-Sung
authors.initials: JS
authors.lastname: Kim
authors.forename: Jin-Hee
authors.initials: JH
authors.lastname: Wang
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Biel
authors.forename: Do-Sung
authors.initials: DS
authors.lastname: Kim
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Flores-Toro
authors.forename: Richa
authors.initials: R
authors.lastname: Vijayvargiya
authors.forename: Ivan
authors.initials: I
authors.lastname: Zendejas
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Behrns
mesh_terms: D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071193:Autophagy-Related Protein 7; D000071186:Beclin-1; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000009:adverse effects*; D002220:Carbamazepine / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D015427:Reperfusion Injury / Q000188:drug therapy
chemical_list: D000927:Anticonvulsants; D051017:Apoptosis Regulatory Proteins; C502661:Atg7 protein, mouse; D000071186:Beclin-1; C115010:Becn1 protein, mouse; D008869:Microtubule-Associated Proteins; D002220:Carbamazepine; D002154:Calpain; D000071193:Autophagy-Related Protein 7; D002118:Calcium
keywords: Atg; Autophagy; CBZ; CQ; Calcium; FDA; Food & Drug Administration; Hepatocytes; I/R; IP(3); Ischemia/reperfusion; KRH; Krebs–Ringer-N-2 hydroxyethylpiperazine-N-2 ethanesulfonic acid; LC3; MPT; Mitochondria; Mitochondrial permeability transition; PI; TMRM; autophagy-related proteins; carbamazepine; chloroquine; inositol-1,4,5-triphosphate; ischemia/reperfusion; microtubule-associated protein 1 light chain 3; mitochondrial permeability transition; propidium iodide; tetramethylrhodamine methylester