Abstract
ABSTRACTObjective: To explore the impact of oxidative insults on mitochondrial dynamics. In mammalian cells, oxidative insults activate stress response pathways including inflammation, cytokine secretion, and apoptosis. Intriguingly, mitochondria are emerging as a sensitive network that may function as an early indicator of subsequent cellular stress responses. Mitochondria form a dynamic network, balancing fusion, mediated by optic atrophy-1 (OPA1), and fission events, mediated by dynamin-related protein-1 (DRP1), to maintain homeostasis.Methods: Here, we examine the impact of oxidative insults on mitochondrial dynamics in 143B osteosarcoma and H9c2 cardiomyoblast cell lines via confocal microscopy, flow cytometry, and protein-based analyses.Results: When challenged with hydrogen peroxide (H2O2), a ROS donor, both cell lines display fragmentation of the mitochondrial network and loss of fusion-active OPA1 isoforms, indicating that OPA1-mediated mitochondrial fusion is disrupted by oxidative damage in mammalian cells. Consistent with this, cells lacking OMA1, a key protease responsible for cleavage of OPA1, are protected against OPA1 cleavage and mitochondrial fragmentation in response to H2O2 challenge.Discussion: Taken together, these findings indicate that oxidative insults damage OPA1-mediated mitochondrial dynamics in mammalian cells via activation of OMA1, consistent with an emerging role for mitochondrial dynamics as an early indicator of cellular stress signaling.Abbreviations: Δψm: transmembrane potential; ROS: reactive oxygen species; H2O2: hydrogen peroxide; OPA1: optic atrophy-1; MFN1: mitofusin1; DRP1: dynamin-related protein 1; DMEM: Dulbecco’s Modified Eagle’s Medium; PBS: phosphate buffer saline; TOM20: translocase of the outer mitochondrial membrane-20; DAPI: diaminophenylindole; TMRE: tetramethylrhodamine ethyl ester; TBST: Tris-Buffered Saline Tween-20; MEF: mouse embryonic fibroblast.
Highlights
Oxidative insults activate critical cellular stress response pathways, with severe outcomes including inflammation, proinflammatory cytokine secretion, and apoptosis
We show that hydrogen peroxide (H2O2)-mediated oxidative stress disrupts mitochondrial dynamics in both H9c2 cardiomyoblasts and 143B osteosarcomas, causing fragmentation of the mitochondrial network and cleavage of fusion-active L-optic atrophy-1 (OPA1) isoforms
Cells lacking the OMA1 metalloprotease are protected from these impacts, retaining L-OPA1 and mitochondrial interconnection under H2O2 challenge
Summary
Oxidative insults activate critical cellular stress response pathways, with severe outcomes including inflammation, proinflammatory cytokine secretion, and apoptosis. Oxidative stressors such as H2O2 cause apoptosis via activation of apoptosis-inducing factor (AIF) and caspase-3 [1,2], resulting in decreased cell viability [3]. H2O2 engages inflammatory signaling, activating the inflammasome via induction of NLRP3 and subsequent secretion of proinflammatory cytokines including IL-1Β [4,5]. Loss of either OPA1 or DRP1 severely disrupts mitochondrial dynamics, and activates inflammatory signaling [12,13] via the NLRP3 inflammasome [4,6]. To explore the impact of oxidative insults on mitochondrial dynamics as a general mechanism, we employ two cell lines, H9c2 cardiomyoblasts and 143B osteosarcomas, with very different origins and metabolic settings
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