Abstract Body (Do not enter title and authors here): Introduction: Ferroptosis, an iron mediated regulated form of cell death has been implicated in myocardial ischemia-reperfusion (IR) injury among other cardiac conditions. Cellular lipid metabolism and lipid oxidation are key processes in ferroptosis. Circadian clocks, transcriptional feedback loops present in all tissues, regulate diurnal variations in various cardiac physiologic processes including the response to IR injury. The degree to which the clock machinery regulates ferroptosis is unknown. We hypothesize that ferroptosis pathways are induced differently in response to ischemia reperfusion injury at different times of day.
Methods: We housed wild-type C57BL6/J mice at a regular 12:12 light dark cycle. We performed a closed chest ischemia reperfusion (IR) procedure to the left anterior descending coronary artery either 2 hours post lights on (Zeitgeber Time 2) or 2 hours post lights off (ZT 14). We sacrificed the mice 24 hours post reperfusion and collected hearts for molecular clock and ferroptosis gene expression quantification by RT-qPCR and to measure a large panel of oxidized lipids and metabolites of oxidation pathways. Because the surgical mice were sacrificed at opposing times of day, we accounted for normal day to night variations by reporting all measurements as the number of standard deviations away from the measurement mean of non-surgical mice sacrificed at the same time of day.
Results: The expression of the anti-ferroptosis molecular clock gene Bmal1 was decreased beyond its normal diurnal variations in the mice with the IR procedure at ZT2 compared to those at ZT14. Furthermore, the expression levels of ferroptosis genes Alox15 and Acsl4 were higher in the ZT2 group compared to ZT14 indicating higher induction of ferroptosis in the ZT2 group beyond that of normal diurnal variation. From our metabolomics panel, we find a higher ratio of NADP+ to NADPH in the ZT 2 group indicating a higher redox burden. Also, acyl carnitines of differing chain lengths showed a consistent decrease in the ZT 2 group.
Conclusions: In response to ischemia reperfusion injury, ferroptosis is induced in a diurnal manner reflective of alterations in the core clock gene Bmal1, lipid metabolism and oxidation pathways.