Activation of mitochondrial‐u‐calpain sensitizes opening of the mitochondrial permeability transition pore during ischemia‐reperfusion (648.11)

Abstract

BACKGROUND: Opening of the mitochondrial permeability transition pore (MPTP) increases cardiac injury during ischemia‐reperfusion (IR). IR activates both cytosolic and mitochondrial calpains. Administration of MDL‐28170 (MDL, a calpain inhibitor) decreases injury in isolated hearts during IR. We asked if inhibition of calpains during IR decreases MPTP opening and if activation of mitochondrial u‐calpain (m‐u‐calpain) predisposes to MPTP.METHODS: Buffer‐perfused mouse hearts (C57BL/6) were subjected to 25 min ischemia and 30 min reperfusion with or without MDL (10 uM) treatment. Opening of MPTP was evaluated by calcium retention capacity (CRC, nmol calcium/mg protein) in mitochondria isolated from IR hearts. In control mitochondria (protease‐purified to remove cytosolic contamination), exogenous calcium was used to activate mito‐u‐calpain. The ADP‐stimulated change in inner membrane potential (ΔΨ) during the transition from state 4 to state 3 respiration (reflected by alteration in TMRM fluorescence arbitrary units, A.U.) was used to assess inner membrane integrity.RESULTS: Inhibition of calpains improved CRC in mitochondria following IR, supporting that calpain activation enhanced MPTP opening. In control mitochondria, exogenous calcium progressively depolarized ΔΨ, whereas inhibition of m‐u‐calpain attenuated the extent of depolarization.CONCLUSION: Inhibition of calpain in purified mitochondria reduces the calcium‐induced permeation of the inner membrane, indicating that activation of m‐u‐calpain likely contributes to MPTP opening and cardiac injury during IR. Isolated hearts LDH release LVDP CRC IR 615±47 13±2 424±50 MDL + IR 383±55* 29±5* 560±16* Isolated mito ΔΨ, TMRM, A.U. Calcium uM 0 50 100 DMSO + mito 0.088±0.009 0.050±0.003†0.013±0.003†MDL+ mito 0.087±0.006 0.066±0.004‡ 0.036±0.013‡ Mean ± SEM; LVDP‐developed pressure mmHg; LDH‐mU/g/min; *p<0.05 vs. IR; †p<0.05 vs. calcium (0 uM); ‡ p<0.05 vs. untreated mitochondria (mito).Grant Funding Source: Supported by Scientist Development Grant from AHA