Cardiovascular Branch, NHLBI, NIH, 10 Center Drive Bethesda, MD, USA.
Center for Molecular Medicine, NHLBI, NIH, Bethesda, MD, USA.
Cardiovasc Res. 2019 Feb 1;115(2):385-394. doi: 10.1093/cvr/cvy218.
Knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice abrogates mitochondrial Ca2+ uptake and permeability transition pore (PTP) opening. However, hearts from global MCU-KO mice are not protected from ischaemic injury. We aimed to investigate whether adaptive alterations occur in cell death signalling pathways in the hearts of global MCU-KO mice.
First, we examined whether cell death may occur via an upregulation in necroptosis in MCU-KO mice. However, our results show that neither RIP1 inhibition nor RIP3 knockout afford protection against ischaemia-reperfusion injury in MCU-KO as in wildtype (WT) hearts, indicating that the lack of protection cannot be explained by upregulation of necroptosis. Instead, we have identified alterations in cyclophilin D (CypD) signalling in MCU-KO hearts. In the presence of a calcium ionophore, MCU-KO mitochondria take up calcium and do undergo PTP opening. Furthermore, PTP opening in MCU-KO mitochondria has a lower calcium retention capacity (CRC), suggesting that the calcium sensitivity of PTP is higher. Phosphoproteomics identified an increase in phosphorylation of CypD-S42 in MCU-KO. We investigated the interaction of CypD with the putative PTP component ATP synthase and identified an approximately 50% increase in this interaction in MCU-KO cardiac mitochondria. Mutation of the novel CypD phosphorylation site S42 to a phosphomimic reduced CRC, increased CypD-ATP synthase interaction by approximately 50%, and increased cell death in comparison to a phospho-resistant mutant.
Taken together these data suggest that MCU-KO mitochondria exhibit an increase in phosphorylation of CypD-S42 which decreases PTP calcium sensitivity thus allowing activation of PTP in the absence of an MCU-mediated increase in matrix calcium.
敲除（KO）小鼠线粒体钙单向转运体（MCU）可阻断线粒体钙摄取和通透性转换孔（PTP）开放。然而，全局 MCU-KO 小鼠的心脏并未免受缺血性损伤的保护。我们旨在研究全局 MCU-KO 小鼠心脏中细胞死亡信号通路是否发生适应性改变。
首先，我们研究了细胞死亡是否可能通过 MCU-KO 小鼠中坏死性凋亡的上调而发生。然而，我们的结果表明，RIP1 抑制或 RIP3 敲除在 MCU-KO 小鼠中不能像在野生型（WT）心脏中那样提供对缺血再灌注损伤的保护，表明缺乏保护作用不能用坏死性凋亡的上调来解释。相反，我们已经确定了 MCU-KO 心脏中环孢菌素 D（CypD）信号的改变。在钙离子载体存在的情况下，MCU-KO 线粒体摄取钙并确实发生 PTP 开放。此外，MCU-KO 线粒体的 PTP 开放具有较低的钙保留能力（CRC），表明 PTP 的钙敏感性更高。磷酸化蛋白质组学鉴定出 MCU-KO 中 CypD-S42 的磷酸化增加。我们研究了 CypD 与假定的 PTP 成分 ATP 合酶的相互作用，并在 MCU-KO 心脏线粒体中鉴定出这种相互作用增加了约 50%。将 CypD 的新磷酸化位点 S42 突变为磷酸模拟物可降低 CRC，增加 CypD-ATP 合酶相互作用约 50%，并与磷酸化抗性突变体相比增加细胞死亡。
综上所述，这些数据表明，MCU-KO 线粒体表现出 CypD-S42 的磷酸化增加，这降低了 PTP 的钙敏感性，从而允许在没有 MCU 介导的基质钙增加的情况下激活 PTP。
