Abstract Background Cardiac ischaemia-reperfusion injury (IRI) is the paradoxical exacerbation of tissue injury following the restoration of blood-flow post myocardial infarction. IRI is initiated by a burst of reactive oxygen species (ROS) during the first few seconds of reperfusion1. ROS are generated by reverse electron transport (RET) via complex I (CI) of the mitochondrial respiratory chain1,2. In order for RET to occur CI must be active3. However, during ischaemia, CI undergoes a structural change known as the active/deactive transition wherein CI assumes a catalytically deactive state4,5. When assessed in vitro, reactivation of CI is associated with a catalytic lag5. Given that active CI is essential to generate mitochondrial ROS immediately upon reperfusion, this work aims to quantify the active population of CI after a period of ischaemia. Methods We establish a spectrophotometric assay to quantify populations of active and deactive CI where enzyme activity is assessed via the rate of NADH oxidation. Alkylation of deactive CI by N-Ethylmaleimide is used to prevent reactivation and therefore active/deactive populations can be determined. A global in situ ischaemia model is used where hearts are sampled at 0, 2, 5, 10, and 30 minutes ischaemia and active/deactive populations assessed. Results Deactivation of CI takes place within the first five minutes of ischaemia with approximately 40% of CI deactivated. No further deactivation is seen after 30 minutes ischaemia. Conclusions A method to quantify active/deactive populations of CI was established and used to demonstrate that 40% of the enzyme is deactivated within the first five minutes of ischaemia in the murine heart. Our findings suggest that, a population of CI remains active after ischaemia and capable of catalysing RET ROS production. Future work is required to determine whether this population will provide sufficient ROS to be responsible for IRI.
Hammond et al. (Fri,) studied this question.