Mitochondrial metabolism relies on membrane potential (ΔΨm) for electron transport across respiratory complexes and a filamentous mitochondrial network. Oxidative metabolism is measured via bioenergetic assays utilizing electron transport chain (ETC) 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 unknown.
Pulmonary hypertension (PH) is characterized by cell hyperproliferation and a metabolic defect; PH-VSMC preferentially use aerobic glycolysis for energy production, experiencing a metabolic switch similar to cancer. Dysfunction in ETC Complex-I has been proposed to play a role in PH etiology, but it’s unclear if this defect is structural or functional.
Mitochondrial metabolism was measured via micropolarimetry using oligomycin, FCCP, Antimycin-A and rotenone in normal and PH patient pulmonary artery smooth muscle cells (hPASMC). The effects of inhibitor treatment on mitochondrial morphology and ΔΨm were determined via continuous collection of data at intervals mirroring classic micropolarimetry. Morphology was assessed via confocal imaging of MitoTracker-Deep-Red-loaded hPASMC with mitochondrial shape/volume calculated with machine learning. ΔΨm was measured by flow-cytometry of tetramethylrhodamine (TMRE)-loaded hPASMC.
Compared to PH, normal hPASMC exhibit significantly higher baseline oxidative metabolism. PH PASMC display mitochondrial fragmentation (~60% intermediate/fragmented); normal PASMC are highly fused (~75% filamentous). ETC inhibitors induce fragmentation in normal PASMC, reducing filamentous network in favor of intermediate rods. Fragmentation is enhanced in ETC-inhibited PH PASMC (>80% fragmented/intermediate). ETC inhibition induces a 10-50% reduction of ΔΨm in normal PASMC. PH PASMC are comparatively hyperpolarized, and largely resistant to inhibitor-induced depolarization.
ETC inhibitors used to study oxidative metabolism have off-target effects in normal VSMC, causing membrane depolarization and fragmentation. In PH, the mitochondrial network is destabilized and sensitive to fragmentation by ETC inhibition, but hyperpolarized and resistant to ETC inhibitor-induced depolarization.