Center for Translational Science, Florida International University, 11350 SW Village Parkway, Port St. Lucie, FL, 34987-2352, USA; Department of Environmental Health Sciences, Robert Stempel College of Public Health and Social Work, Florida International University, Miami, FL, 33199, USA.
Center for Translational Science, Florida International University, 11350 SW Village Parkway, Port St. Lucie, FL, 34987-2352, USA.
Free Radic Biol Med. 2024 Jan;210:183-194. doi: 10.1016/j.freeradbiomed.2023.11.008. Epub 2023 Nov 17.
Pulmonary hypertension (PH) is a progressive disease with vascular remodeling as a critical structural alteration. We have previously shown that metabolic reprogramming is an early initiating mechanism in animal models of PH. This metabolic dysregulation has been linked to remodeling the mitochondrial network to favor fission. However, whether the mitochondrial fission/fusion balance underlies the metabolic reprogramming found early in PH development is unknown.
Utilizing a rat early model of PH, in conjunction with cultured pulmonary endothelial cells (PECs), we utilized metabolic flux assays, Seahorse Bioassays, measurements of electron transport chain (ETC) complex activity, fluorescent microscopy, and molecular approaches to investigate the link between the disruption of mitochondrial dynamics and the early metabolic changes that occur in PH.
We observed increased fusion mediators, including Mfn1, Mfn2, and Opa1, and unchanged fission mediators, including Drp1 and Fis1, in a two-week monocrotaline-induced PH animal model (early-stage PH). We were able to establish a connection between increases in fusion mediator Mfn1 and metabolic reprogramming. Using an adenoviral expression system to enhance Mfn1 levels in pulmonary endothelial cells and utilizing C-glucose labeled substrate, we found increased production of C lactate and decreased TCA cycle metabolites, revealing a Warburg phenotype. The use of a C-glutamine substrate showed evidence that hyperfusion also induces oxidative carboxylation. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels secondary to the disruption of cellular bioenergetics and higher levels of mitochondrial reactive oxygen species (mt-ROS). The elevation in mt-ROS correlated with attenuated ETC complexes I and III activities. Utilizing a mitochondrial-targeted antioxidant to suppress mt-ROS, limited HIF-1α protein levels, which reduced cellular glycolysis and reestablished mitochondrial membrane potential.
Our data connects mitochondrial fusion-mediated mt-ROS to the Warburg phenotype in early-stage PH development.
肺动脉高压（PH）是一种进行性疾病，其血管重构是一种关键的结构改变。我们之前已经表明，代谢重编程是 PH 动物模型中的早期起始机制。这种代谢失调与重塑线粒体网络以促进裂变有关。然而，在 PH 早期发展中发现的代谢重编程是否依赖于线粒体裂变/融合平衡尚不清楚。
利用 PH 的大鼠早期模型，结合培养的肺内皮细胞（PECs），我们利用代谢通量测定、 Seahorse 生物测定、电子传递链（ETC）复合物活性测量、荧光显微镜和分子方法来研究线粒体动力学的破坏与 PH 中发生的早期代谢变化之间的联系。
我们观察到融合介体（包括 Mfn1、Mfn2 和 Opa1）增加，而分裂介体（包括 Drp1 和 Fis1）不变，在两周的单克隆毒素诱导的 PH 动物模型（早期 PH）中。我们能够建立融合介体 Mfn1 增加与代谢重编程之间的联系。使用腺病毒表达系统增强肺内皮细胞中的 Mfn1 水平，并利用 C-葡萄糖标记的底物，我们发现 C-乳酸产量增加，三羧酸循环代谢物减少，揭示了沃伯格表型。使用 C-谷氨酰胺底物表明，超融合还会诱导氧化羧化作用。糖酵解的增加与细胞生物能量学的破坏和线粒体活性氧物质（mt-ROS）水平升高导致的缺氧诱导因子 1α（HIF-1α）蛋白水平升高有关。mt-ROS 的增加与 ETC 复合物 I 和 III 活性的降低相关。利用线粒体靶向抗氧化剂抑制 mt-ROS，可降低 HIF-1α 蛋白水平，减少细胞糖酵解并重建线粒体膜电位。
我们的数据将线粒体融合介导的 mt-ROS 与早期 PH 发展中的沃伯格表型联系起来。
