Pulmonary arterial hypertension (PAH) is a pathological state characterized by elevated pulmonary arterial pressure (PAP) and increased pulmonary vascular resistance (PVR). A key feature of PAH is hyperproliferation and apoptosis-resistance in pulmonary arterial smooth muscle cell (PASMC). This phenotype is related in part to mitochondrial abnormalities including increased fragmentation and a shift to aerobic glycolysis. However, we have noted altered mitochondrial motility in PAH as well. Higher mitochondrial motility is associated with increased cell proliferation in a previous study.
Mitochondria from PAH and normal PASMC were imaged by loading with tetramethylrhodamine methyl ester perchlorate (TMRM), a mitochondrial-targeted, potentiometric dye. High resolution Z-stack and time-lapse imaging were performed using a Leica 3D STED super resolution confocal microscope. Size (μm3), shape (sphericity) and position of every mitochondrion were tracked every 5 seconds for 5 minutes and measured with LAS-X software (Leica). Mean velocity of each mitochondrion was measured as total distance (nm)/total time (second) tracked by Imaris software (Bitplane). Expression levels of mitochondrial motility-related proteins (Miro, Milton, kinesin and dynein) in whole cell lysate were quantified by western blot analyses using ß-actin as internal control.
Mitochondrial velocity is significantly increased in PAH PASMCs compared to normal PASMCs (61.1±1.8 nm/s vs 52.9±1.7 nm/s vs p=0.0009). A higher percentage of mitochondria are moving at a high speed (>100nm/s)in PAH PASMCs (28.5% in PAH vs 20.4% in normal). There is no significant change in the expression levels of Miro, Milton, kinesin and dynein in PAH PASMCs.
Mitochondrial are fragmented and their motility is significantly increased in PAH PASMCs. Mitochondrial motility-related proteins are largely unchanged in PAH PASMCs. We next intend to investigate functional changes in the calcium sensor in Miro and potential alteration in GTPase activity as a basis for dysregulated motility