Background:
To date, there are no FDA-approved therapies for the reduction of infarct size in acute myocardial infarction. Previously, we developed a cell-based phenotypic assay of ischemia-reperfusion (IR) injury, which was used to identify novel cytoprotective agents delivered prior to ischemia. Herein, we sought to identify cytoprotective agents in a more clinically relevant model: drug delivery at reperfusion, and to investigate possible underlying mechanisms of protection.
Methods:
Primary adult mouse cardiomyocytes were subjected to simulated IR injury using a modified Seahorse XF24 apparatus with drug addition at the onset of reperfusion. Cell death was estimated using LDH release. Drugs which protected cardiomyocytes in vitro were tested in a Langendorff model of IR injury, measuring functional recovery and infarct size. In separate experiments, metabolites extracted from perfused hearts were resolved by HPLC.
Results:
Nornicotine was identified as a cardioprotective agent in the screen. In perfused hearts, 10 nM nornicotine injected at the onset of reperfusion improved functional recovery and decreased in infarct size (13.1% ± 2.4 vs 49.2% ± 2.5 in non-treated hearts, p<0.05, n=16-20). Nornicotine also exhibited profound inhibitory effects on mitochondrial complex I activity.
Succinate is known to accumulate in ischemia, and its rapid consumption during early reperfusion exacerbates reperfusion injury via ROS generation from electron backflow through complex I [PMID: 25383517]. In non-treated hearts, we confirmed that high post ischemic levels of succinate rapidly declined during the first 2 min of reperfusion. In contrast, nornicotine slowed post-ischemic succinate consumption, suggesting that electron backflow through complex I is the major pathway driving succinate consumption.
Conclusions:
Herein, we demonstrated that nornicotine was cardioprotective when delivered at early reperfusion in vitro and ex vivo. The mechanism of cardioprotection may be due to inhibition of rapid succinate consumption during early reperfusion via reverse electron flow back through complex I.
GW28-e0866 Partial loss of NDUFA13 in mitochondrial complex I attenuates ischemia-reperfusion injury
