10 - Electron transport chain inhibitors alter membrane potential and induce mitochondrial fragmentation in vascular smooth muscle cells: implications for bioenergetic measurements

Abstract

Mitochondrial metabolism relies on membrane potential (ΔΨm) for effective electron transport across respiratory complexes and a filamentous mitochondrial network. Oxidative metabolism is typically measured via bioenergetic assays utilizing electron transport chain inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor). Bioenergetic effects of these compounds are well-established, but effects on ΔΨm and morphology in vascular smooth muscle cells (VSMC) remains largely unknown. Pulmonary hypertension (PH) is complex vasculopathy characterized by excess cell proliferation and a metabolic defect – PH VSMC preferentially use aerobic glycolysis for energy production, having experienced a metabolic switch similar to other proliferative diseases, including cancer. Using normal and PH patient pulmonary artery smooth muscle cells (hPASMC), mitochondrial metabolism was measured via micropolarimetry using oligomycin, FCCP, Antimycin A and rotenone. Mitochondrial morphology was assessed pre- and post-inhibitor treatment by confocal imaging of MitoTracker Deep Red-loaded hPASMC, and subsequent application of a machine learning algorithm to predict mitochondrial shape and calculate volume. ΔΨm was measured pre- and post- inhibitor treatment by flow cytometry of tetramethylrhodamine (TMRE)-loaded hPASMC. Compared to PH, normal hPASMC exhibit significantly higher baseline oxidative metabolism. PH PASMC display significant mitochondrial fragmentation (~60% intermediate/fragmented); normal PASMC are highly fused (~75% filamentous). ETC inhibitors induce fragmentation in normal PASMC, significantly reducing filamentous network in favor of intermediate rods. Fragmentation is further enhanced in ETC-inhibited PH PASMC (>80% fragmented/intermediate). ETC inhibitors also induce a 10-50% reduction of ΔΨm in normal PASMC. PH PASMC are comparatively hyperpolarized, and largely resistant to inhibitor-induced depolarization, suggesting the underlying metabolic defect in PH PASMC is independent of ΔΨm. ETC inhibitors used to study oxidative metabolism have off-target effects in normal VSMC, causing membrane depolarization and fragmentation. In PH patients, the mitochondrial network is destabilized and sensitive to further fragmentation by ETC inhibition, but this fragmentation is independent of membrane potential.