Cardiomyocyte derived extracellular mitochondria in activation of macrophages during oxidative stress

Mitochondria serve as central hubs for aerobic metabolism and regulators of cell fate. Damage to mitochondria induced by oxidative stress contributes to cardiac injury. We investigate whether oxidative stress causes the release of mitochondria into the extracellular space and whether these extracellular mitochondria (EM) mediate the detrimental effect of oxidative stress.

AC16 cardiomyocytes were exposed to sublethal doses of H2O2 to collect mitochondria released into the conditioned culture medium. These EM were compared with intracellular mitochondria (IM) for morphology, size, membrane potential, and metabolic profile using liquid chromatography-tandem mass spectrometry-based metabolomics. Cellular ceramide content was measured by lipidomics to determine the role of ceramide synthesis in mitochondrial release. EM were tested for the ability to activate THP-1 macrophages. Oxidants caused an increase in EM. While EM from stressed cells did not show a significant difference from those of non-stressed cells in overall morphology, size, or surface charge, EM exhibited disrupted cristae structure, smaller size, reduced membrane potential, and decreased levels of nicotinamide adenine dinucleotide, adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate compared to IM. H2O2 treatment upregulated several ceramide species in AC16 cells and the inhibition of ceramide synthesis markedly reduced EM release under oxidative stress. Functionally, EM activated M1 and M2 like macrophages, as indicated by increased expression of the cytokine markers tumour necrosis factor α and CD163.

Oxidative stress enhanced the release of mitochondria from cardiomyocytes into the extracellular space. These EM differ from IM in their smaller sizes, reduced membrane potential, and depressed metabolic state. At high abundance, EM act as mediators that promote macrophage activation.