Abstract
Objective:
Cell-free hemoglobin (CFH) is released into the circulation during sepsis where it can redox cycle from the ferrous 2+ to ferric 3+ and disrupt endothelial function, but the mechanisms of CF-mediated endothelial dysfunction are unknown. We hypothesized that oxidized CFH induces mitochondrial dysfunction via the mitochondrial permeability transition pore (mPTP) in pulmonary endothelial cells, leading to the release of mitochondrial DNA (mtDNA).
Methods:
Human lung microvascular endothelial cells were treated with CFH2+/CFH3+. We measured mitochondrial mPTP activation (flow cytometry), network and mass (immunostaining), structure (electron microscopy), mtDNA release (PCR), and oxygen consumption rate (OCR; Seahorse). Plasma from critically ill patients and conditioned cell media were quantified for mtDNA and CFH.
Results:
CFH3+ disrupted the mitochondrial network, activated the mPTP (1434 (874-1642) vs. 2302 (1729-2654) mean fluorescent intensity, p = 0.02), increased the spare respiratory capacity (30.61 (29.36-37.78) vs. 7.83 (3.715-10.63) OCR, p = 0.004), and caused the release of mtDNA. CFH was associated with circulating mtDNA (R2 = 0.1912, p = 0.0077) in plasma from critically ill patients.
Conclusion:
CFH3+, not CFH2+, is the primary driver of CFH-induced lung microvascular mitochondrial dysfunction. Activation of the mPTP and the release of mtDNA are a feature of CFH3+ mediated injury.
Keywords:
cell‐free hemoglobin; mitochondrial dysfunction; mitochondrial permeability transition pore; mtDNA.