Anesthetic-induced Preconditioning Delays Opening of Mitochondrial Permeability Transition Pore via  Protein Kinase C-ϵ–mediated Pathway

Abstract

Background Cardioprotection by volatile anesthetic-induced preconditioning (APC) involves activation of protein kinase C (PKC). The current study investigated the importance of APC-activated PKC in delaying mitochondrial permeability transition pore (mPTP) opening. Methods Rat ventricular myocytes were exposed to isoflurane in the presence or absence of nonselective PKC inhibitor chelerythrine or isoform-specific inhibitors of PKC-δ (rottlerin) and PKC-ε (myristoylated PKC-ε V1-2 peptide), and the mPTP opening time was measured using confocal microscopy. Ca2+-induced mPTP opening was measured in mitochondria isolated from rats exposed to isoflurane in the presence and absence of chelerythrine, or in mitochondria directly treated with isoflurane after isolation. Translocation of PKC-ε was assessed in APC and control cardiomyocytes by Western blotting. Results In cardiomyocytes, APC prolonged time necessary to induce mPTP opening (261±26 s APC vs. 216±27 s control; P<0.05), while chelerythrine abolished this delay to 213±22 s. The effect of isoflurane was also abolished when PKC-ε inhibitor was applied (210±22 s), but not in the presence of PKC-δ inhibitor (269±31 s). Western blotting revealed translocation of PKC-ε toward mitochondria in APC cells. The Ca2+ concentration required for mPTP opening was significantly higher in mitochondria from APC rats (45±8 μM mg-1 control vs. 64±8 μM mg-1 APC), and APC effect was reversed with chelerythrine. In contrast, isoflurane did not protect directly treated mitochondria. Conclusion APC induces delay of mPTP opening through PKC-ε-mediated inhibition of mPTP opening, but not through PKC-δ. These results point to the connection between cytosolic and mitochondrial components of cardioprotection by isoflurane.

Catestatin reduces myocardial ischaemia/reperfusion injury: involvement of PI3K/Akt, PKCs, mitochondrial KATP channels and ROS signalling

“…The cardiac activity of CST is achieved through signalling pathways expected for receptor-orphan peptides with membraneinteracting properties [4]. Here, using classic antagonists [14,35,39,[41][42][43][44], we demonstrated for the first time that the cardioprotective effect of CST-Post depends on the activation of PI3k/Akt, PKCs and of mitoK ATP channels, which may include a ROS signalling. The prevention of mPTP opening in myocytes challenged with oxidative stress reinforces the idea that CST protective effects converge on mitochondria.…”

“…In Group 5 a specific PKCε inhibitor, εV1-2, was used to test the involvement of this kinase (εV1-2 1μM; CST-Post+εV1-2 Group) [44].…”

Mitochondrial depolarization underlies delay in permeability transition by preconditioning with isoflurane: roles of ROS and Ca2+

“…H 2O2 was not used to trigger mPTP opening, since it caused cell hypercontracture, which hampered identification of pore opening. Instead, mPTP opening was instigated using a well-established approach, where oxidative stress was induced by laser-photoexcitation as described below and previously (42). H 2O2 alone did not affect fluorescence probes [except 5-(and-6)-chloromethyl-2=,7=-dichlorofluorescein (CM-DCF)], as measured by spectrofluorometry.…”

“…Cardiac mitochondria were isolated from Wistar rats as reported previously (42). Hearts were placed in cold isolation buffer [in mM: 50 sucrose, 200 mannitol, 5 KH 2PO4, 1 EGTA, 5 3-(N-morpholino) propanesulfonic acid, and 0.1% bovine serum albumin; pH 7.3 adjusted with KOH] and homogenized with a T 25 disperser (IKA-Werke, Staufen, Germany).…”

Direct evidence for inhibition of mitochondrial permeability transition pore opening by sevoflurane preconditioning in cardiomyocytes: Comparison with cyclosporine A

“…Recent studies have found that the volatile anesthetics such as isoflurane and sevoflurane induce cardioprotection by preconditioning and postconditioning (Bouwman et al, 2010;Inamura et al, 2010;Inamura et al, 2009;Kaneda et al, 2008;Okusa et al, 2009;Weber et al, 2005). Pravdic et al demonstrated that isoflurane preconditioning delays MPTP opening using the tetramethyl-rhodamine ethyl ester (TMRE) technique in rat cardiomyocytes (Pravdic et al, 2009). Also, it has been reported that atractyloside, a MPTP opener, abolished cardioprotection by isoflurane postconditioning (Krolikowski et al, 2005).…”