Cytochrome c Rescue of Mitochondrial Function

Cytochrome c is an important protein of the respiratory chain and loss can lead to reduction in respiratory activity. Exogenous cytochrome c addition may rescue mitochondrial function (23 (link), 24 (link)). Therefore, permeabilized HL-1 cardiomyocytes were measured via Seahorse XFe96 Flux Analyzer after pre-incubation with IPA for 24 h as previously described. 1 mM exogenous cytochrome c (cytochrome c from equine heart; final concentration: 100 μM) was simultaneously injected with saponin (final concentration of 250 μg/ml), 4 mM ADP, 10 mM succinate, and 2 μM rotenone. After two baseline measurements, respiratory activity was measured five times with cycles of mix (0.5 min)/wait (0.5 min)/measure (2 min). At the end of the measurement a mix of 20 μM rotenone/antimycin A (final concentration: 2 μM) was injected to the cells.