School of Biochemistry and Bristol Heart Institute, University of Bristol, Bristol, United Kingdom.
Centre for Synaptic Plasticity, School of Biochemistry, University of Bristol, Bristol, United Kingdom.
PLoS One. 2020 Jun 24;15(6):e0234653. doi: 10.1371/journal.pone.0234653. eCollection 2020.
We previously demonstrated that hexokinase II (HK2) dissociation from mitochondria during cardiac ischemia correlates with cytochrome c (cyt-c) loss, oxidative stress and subsequent reperfusion injury. However, whether HK2 release is the primary signal mediating this ischemia-induced mitochondrial dysfunction was not established. To investigate this, we studied the effects of dissociating HK2 from isolated heart mitochondria. Mitochondria isolated from Langendorff-perfused rat hearts before and after 30 min global ischemia ± ischemic preconditioning (IPC) were subject to in vitro dissociation of HK2 by incubation with glucose-6-phosphate at pH 6.3. Prior HK2 dissociation from pre- or end-ischemic heart mitochondria had no effect on their cyt-c release, respiration (± ADP) or mitochondrial permeability transition pore (mPTP) opening. Inner mitochondrial membrane morphology was assessed indirectly by monitoring changes in light scattering (LS) and confirmed by transmission electron microscopy. Although no major ultrastructure differences were detected between pre- and end-ischemia mitochondria, the amplitude of changes in LS was reduced in the latter. This was prevented by IPC but not mimicked in vitro by HK2 dissociation. We also observed more Drp1, a mitochondrial fission protein, in end-ischemia mitochondria. IPC failed to prevent this increase but did decrease mitochondrial-associated dynamin 2. In vitro HK2 dissociation alone cannot replicate ischemia-induced effects on mitochondrial function implying that in vivo dissociation of HK2 modulates end-ischemia mitochondrial function indirectly perhaps involving interaction with mitochondrial fission proteins. The resulting changes in mitochondrial morphology and cristae structure would destabilize outer / inner membrane interactions, increase cyt-c release and enhance mPTP sensitivity to [Ca2+].
我们之前的研究表明，在心脏缺血期间，己糖激酶 II（HK2）与线粒体分离与细胞色素 c（cyt-c）的丢失、氧化应激以及随后的再灌注损伤有关。然而，HK2 的释放是否是介导这种缺血诱导的线粒体功能障碍的主要信号尚未确定。为了研究这一点，我们研究了分离的心脏线粒体中 HK2 分离的影响。在 Langendorff 灌注大鼠心脏缺血前和缺血 30 分钟后（缺血预处理）分离的线粒体，通过在 pH 6.3 下用葡萄糖-6-磷酸孵育，使 HK2 与线粒体分离。预先或末期缺血心脏线粒体中 HK2 的分离对它们的 cyt-c 释放、呼吸（±ADP）或线粒体通透性转换孔（mPTP）开放没有影响。通过监测光散射（LS）的变化间接评估线粒体内膜形态，并通过透射电子显微镜证实。尽管在缺血前和末期缺血线粒体之间没有检测到主要的超微结构差异，但后者 LS 变化的幅度减小。IPC 可防止这种变化，但 HK2 分离在体外不能模拟这种变化。我们还观察到末期缺血线粒体中更多的分裂蛋白 Drp1。IPC 未能阻止这种增加，但确实减少了与线粒体相关的动力蛋白 2。HK2 分离本身不能复制缺血对线粒体功能的诱导作用，这表明体内 HK2 的分离可能通过与线粒体分裂蛋白的相互作用间接调节末期缺血线粒体的功能。线粒体形态和嵴结构的变化会破坏外膜/内膜相互作用，增加 cyt-c 的释放，并增强 mPTP 对 [Ca2+]的敏感性。
