OBJECTIVE L5, a highly electronegative subtype of low-density lipoprotein (LDL), is likely associated with the development of atherosclerosis and cardiovascular diseases. Normal LDL is composed mainly of apolipoprotein (Apo) B, but L5 has additional proteins such as ApoE. We previously demonstrated that L5 induces endothelial cell senescence by increasing mitochondrial reactive oxygen species. In the present study, we examined the effect of L5 on mitochondrial function in cardiomyocytes. METHODS We used the Seahorse XF24 extracellular flux analyzer to examine the effect of L5 and its components on mitochondrial energy production. The effects of L5 on mitochondrial morphology were examined by immunofluorescence using MitoTracker Green FM and the corresponding probes in H9c2 cardiomyoblasts. Mitochondrial permeability was assessed by using a calcium-induced swelling assay with a voltage-dependent anion-selective channel (VDAC) inhibitor to determine VDAC-dependence both in vitro and in vivo. L5 without ApoE, referred to as △L5, was used to clarify the role of ApoE in L5-induced mitochondrial dysfunction. RESULTS L5 not only significantly decreased basal (P < 0.05) and maximal respiration (P < 0.01) but also reduced spare respiratory capacity (P < 0.01) in H9c2 cells. Additionally, L5 caused phosphorylation of Drp1 and mitochondrial fission. Recombinant ApoE mimicked the mitochondrial effects of L5, but △L5 did not cause similar effects. After entering cells, ApoE on L5 colocalized with mitochondrial VDAC and caused mitochondria swelling both in vitro and in vivo. This effect was also seen with recombinant ApoE but not △L5. CONCLUSIONS ApoE may play an important role in electronegative LDL-induced mitochondrial dysfunction through the opening of the mitochondrial permeability transition pore via the interaction of ApoE and VDAC.

中文翻译：

---

载脂蛋白E在心肌细胞负电性低密度脂蛋白诱导的线粒体功能障碍中的作用。

目的L5是低密度脂蛋白（LDL）的高度负电性亚型，可能与动脉粥样硬化和心血管疾病的发展有关。正常的LDL主要由载脂蛋白（Apo）B组成，但L5具有其他蛋白，例如ApoE。我们先前证明，L5通过增加线粒体活性氧来诱导内皮细胞衰老。在本研究中，我们检查了L5对心肌细胞线粒体功能的影响。方法我们使用Seahorse XF24细胞外通量分析仪检查L5及其组分对线粒体能量产生的影响。使用MitoTracker Green FM和H9c2心肌母细胞中的相应探针，通过免疫荧光检查了L5对线粒体形态的影响。线粒体通透性通过使用钙诱导的溶胀试验与电压依赖性阴离子选择性通道（VDAC）抑制剂进行评估，以确定体内和体外的VDAC依赖性。没有ApoE的L5（称为△L5）用于阐明ApoE在L5诱导的线粒体功能障碍中的作用。结果L5不仅显着降低H9c2细胞的基础（P <0.05）和最大呼吸（P <0.01），而且降低了备用呼吸能力（P <0.01）。另外，L5引起Drp1的磷酸化和线粒体裂变。重组ApoE模仿了L5的线粒体作用，但△L5没有引起类似的作用。进入细胞后，L5上的ApoE与线粒体VDAC共定位，导致线粒体在体内和体外均发生肿胀。用重组ApoE也可以看到这种效果，但△L5却没有。