From INSERM UMR-1060, CarMeN Laboratory, Université Lyon-1, Faculté de Médecine Rockefeller, Lyon, France (G.T., P.C., M.A., E.C.-L., R.H., B.P., Y.T., A.G., M.O.); Service d'Anesthésie Réanimation, Hôpital Louis Pradel, Hospices Civils de Lyon, Lyon, France (P.C.); INSERM UMR-1046, Université Montpellier 1, Université Montpellier 2, Centre Hospitalier Universitaire de Montpellier, Montpellier, France (J.F., A.L.); and Service d'Explorations Fonctionnelles Cardiovasculaires and CIC de Lyon, Hôpital Louis Pradel, Hospices Civils de Lyon, Lyon, France (M.O.).
Anesthesiology. 2015 Dec;123(6):1374-84. doi: 10.1097/ALN.0000000000000876.
The mitochondrial permeability transition pore (PTP) has been established as an important mediator of ischemia-reperfusion-induced cell death. The matrix protein cyclophilin D (CypD) is the best known regulator of PTP opening. Therefore, the authors hypothesized that isoflurane, by inhibiting the respiratory chain complex I, another regulator of PTP, might reinforce the myocardial protection afforded by CypD inhibition.
Adult mouse or isolated cardiomyocytes from wild-type or CypD knockout (CypD-KO) mice were subjected to ischemia or hypoxia followed by reperfusion or reoxygenation. Infarct size was assessed in vivo. Mitochondrial membrane potential and PTP opening were assessed using tetramethylrhodamine methyl ester perchlorate and calcein-cobalt fluorescence, respectively. Fluo-4 AM and rhod-2 AM staining allowed the measurement, by confocal microscopy, of Ca transient and Ca transfer from sarcoplasmic reticulum (SR) to mitochondria after caffeine stimulation.
Both inhibition of CypD and isoflurane significantly reduced infarct size (-50 and -37%, respectively) and delayed PTP opening (+63% each). Their combination had no additive effect (n = 6/group). CypD-KO mice displayed endogenous protection against ischemia-reperfusion. Isoflurane depolarized the mitochondrial membrane (-28%, n = 5), decreased oxidative phosphorylation (-59%, n = 5), and blunted the caffeine-induced Ca transfer from SR to mitochondria (-22%, n = 7) in the cardiomyocytes of wild-type mice. Importantly, this transfer was spontaneously decreased in the cardiomyocytes of CypD-KO mice (-25%, n = 4 to 5).
The results suggest that the partial inhibitory effect of isoflurane on respiratory complex I is insufficient to afford a synergy to CypD-induced protection. Isoflurane attenuates the Ca transfer from SR to mitochondria, which is also the prominent role of CypD, and finally prevents PTP opening.
线粒体通透性转换孔（PTP）已被确立为缺血再灌注诱导细胞死亡的重要介质。基质蛋白亲环素 D（CypD）是 PTP 开放的最佳已知调节剂。因此，作者假设，异氟烷通过抑制呼吸链复合物 I（另一种 PTP 调节剂），可能会增强 CypD 抑制所提供的心肌保护作用。
成年小鼠或来自野生型或 CypD 敲除（CypD-KO）小鼠的分离心肌细胞接受缺血或缺氧，随后进行再灌注或再氧合。在体内评估梗死面积。使用四甲基罗丹明甲酯过氯酸盐和钙黄绿素-钴荧光分别评估线粒体膜电位和 PTP 开放。通过共焦显微镜用 Fluo-4 AM 和 rhod-2 AM 染色，可以测量咖啡因刺激后 Ca 瞬变和 Ca 从肌浆网（SR）向线粒体的转移。
CypD 抑制和异氟烷均显著减少梗死面积（分别减少 50%和 37%）并延迟 PTP 开放（各增加 63%）。它们的组合没有相加作用（每组 n = 6）。CypD-KO 小鼠对缺血再灌注表现出内源性保护作用。异氟烷使线粒体膜去极化（-28%，n = 5），降低氧化磷酸化（-59%，n = 5），并减弱了野生型小鼠心肌细胞中 SR 向线粒体的咖啡因诱导 Ca 转移（-22%，n = 7）。重要的是，这种转移在 CypD-KO 小鼠的心肌细胞中自发减少（-25%，n = 4 至 5）。
结果表明，异氟烷对呼吸复合物 I 的部分抑制作用不足以提供与 CypD 诱导的保护作用协同作用。异氟烷减弱了 Ca 从 SR 向线粒体的转移，这也是 CypD 的主要作用，最终阻止了 PTP 的开放。
