S105 Paracrine-mediated transfer of mitochondria between airway smooth muscle cells

BackgroundMitochondria are cytoplasmic organelles which produce energy in the form of adenosine triphosphate (ATP) through oxidative phosphorylation. There is evidence of mitochondrial dysfunction in the airway smooth muscle cells (ASMCs) of patients with Chronic Obstructive Pulmonary Disease (COPD). This may contribute to the ASMC hypertrophy and/or hyperplasia observed in COPD lungs. Mitochondrial dysfunction includes decreased ATP production and increased production of mitochondrial reactive oxygen species. Transfer of mitochondria through tunnelling nanotubules (TNTs) and extracellular vesicles (EVs) has been demonstrated between various cell types and has beneficial effects including reducing aspects of mitochondrial dysfunction. Previously, our group reported mitochondrial transfer between ASMCs through TNTs. Therefore, I hypothesised that paracrine-mediated mitochondrial transfer also occurs between ASMCs via EVs.MethodsPrimary human ASMCs from healthy ex-smokers were cultured. Donor cell mitochondria were either stained with MitoTracker Green or transfected with Cell-Light™ Mitochondria-green fluorescent protein and left to secrete mitochondria into the culture medium overnight. The conditioned media (CdM) was collected and transferred onto the recipient cells. The percentage of MitoTracker-positive recipient cells was quantified using flow cytometry. The recipient cells which received CdM from Cell-Light™-transfected donor cells were fixed and imaged with widefield microscopy.ResultsA higher percentage of recipient cells were MitoTracker-positive when incubated overnight with CdM from donor cells compared to control (cells that did not receive CdM; 19.17% ± 7.08 vs 0.74% ± 0.36). Decreasing the donor cell secretion time to four hours led to a decrease in transfer (6.25% ± 4.10) compared to donors which secreted overnight (19.17% ± 7.08). However, decreasing the recipient uptake time to 4 hours did not affect the percentage of MitoTracker-positive cells (18.16% ± 6.02 vs 19.17% ± 7.08).The recipient cells showed green fluorescence after incubation with CdM from Cell-Light™-transfected donor cells, demonstrating genuine transfer of mitochondria from the CdM.ConclusionParacrine-mediated mitochondrial transfer was demonstrated between ASMCs and was affected by donor cell secretion time. Transfection of donor cells with CellLight™ further confirmed paracrine-mediated transfer of mitochondria between ASMCs. Further work to characterise the EVs in the Cd