Role of CLIC proteins in mitochondrial dysfunction in pulmonary arterial hypertension

Abstract

Background: Pulmonary arterial hypertension (PAH) is a severe and currently incurable disease characterised by progressive pulmonary vascular remodelling and right heart hypertrophy. PAH bears many hallmarks of cancer, including apoptosis resistance, increased cell proliferation and mitochondrial dysfunction characterised by membrane hyperpolarization, mitochondrial fission and metabolic switch from mitochondrial oxidative phosphorylation to glycolysis. Chloride intracellular channel proteins CLIC1 and CLIC4 are highly expressed in the remodelled pulmonary vasculature in PAH, and these redox-sensitive proteins regulate cell proliferation and angiogenesis. New data suggests that CLIC1, CLIC4 and CLIC5 may also act as structural components of mitochondrial membranes but their role has not been investigated. Hypothesis: Increased expression of CLIC proteins induces mitochondrial dysfunction in human pulmonary vascular endothelial cells (HPAECs) in vitro, consistent with pathological changes seen in PAH. Methods: CLIC1, CLIC4 and CLIC5 expression was manipulated by adenoviral gene transfer. The effects of CLIC expression changes on mitochondrial fission/fusion, mitochondrial membrane potential (Δψm), mitochondrial reactive oxygen species (mtROS) generation, expression of mitochondrial proteins, glycolysis and mitochondrial respiration were studied in human pulmonary artery endothelial cells (HPAECs) and endothelial colony forming cells (ECFCs) derived from PAH patients and healthy individuals. The subcellular localization of CLIC proteins and their effect on mitochondrial structure were studied using transmission electron microscopy. Mitochondrial content, mitochondrial fragmentation, mitochondrial membrane potential, microtubule dynamics and microtubule-mitochondrial interaction were studied using immunofluorescence and confocal microscopy. Fluorescence-activated cell sorting (FACS) scans were used to detect changes in mitochondrial reactive oxygen species (ROS) generation, while the expression levels of proteins regulating mitochondrial dynamics were analysed by electrophoresis and western blotting. Agilent Seahorse bioenergetics assays were carried out in order to evaluate the potential contribution of CLIC proteins to the Warburg effect. Mitochondrial proteomic analysis of HPAECs overexpressing CLIC proteins was used to identify key signalling pathways. Finally, the effect of CLIC1 and CLIC4 silencing and mitofusin-2 (MFN2) overexpression on CLIC-induced mitochondrial dysfunction in HPAECs, and ECFCs was also investigated. Results: CLIC1 and CLIC4 overexpression induced mitochondrial fission, and this effect was accompanied by a significant decrease in the expression of fission regulators MFN2 and optic atrophy 1 (OPA1) and the increased activation of fission regulator DRP1. Mitochondrial fragmentation was accompanied by changes in the ultrastructure of mitochondrial cristae and the reorganization of microtubule cytoskeleton. CLIC proteins significantly reduced mitochondrial respiration, increased mitochondrial membrane potential and mitochondrial reactive oxygen species (ROS) generation and stimulated glycolysis in HPAECs. These changes were partially prevented by the overexpression of MFN2. Endothelial cells from PAH patients showed increased expression of CLIC1 and CLIC4 accompanied by mitochondrial fragmentation, increased mitochondrial membrane potential and reduced expression of MFN2. The disease phenotype was reversed by CLIC1 and CLIC4 silencing or overexpression of MFN2. Conclusion: CLIC proteins are likely to play a contributory role in mitochondrial dysfunction in PAH.