Rapid regulation of mitochondrial fission and fusion mediators Drp1 and Opa1 during ischemia/reperfusion injury and cardioprotection by ischemic preconditioning

Mitochondria are both, target and culprit of myocardial ischemia and reperfusion (IR) injury. Therefore, mitochondria are critical to all cardioprotective strategies - all known cardioprotective signaling pathways converge at the level of mitochondria. Among different mitochondrial functions, mitochondrial dynamics - regulated by several fission and fusion mediators (e.g. dynamin-related protein 1 (Drp1) and optic atrophy 1 (Opa1)) - seems to be associated with cardioprotection. Knock-down of the fission protein Drp1 reduced infarct size in mice (PLoS One. 2021 Mar 25;16(3):e0248554) and several experimental studies on IR in rodents identified changes in mitochondrial fission and fusion mediators at late reperfusion (≥30 min; e.g. Biomed Res Int. 2022 Sep 27:2022:6889278). However, recent data of non-cardiac tissue highlight a relevance of Drp1 and Opa1 not only for mitochondrial dynamics but also for mitochondrial respiration (Sci Adv. 2024 Apr 5;10(14):eadl0389; Cell. 2013 Sep 26;155(1):160–171). Thus, we here aimed to characterize changes in expression/activation of fission and fusion proteins, in particular Drp1, mitofusin (Mfn)1/2 and Opa1, at different crucial time windows for cardioprotection (before and during ischemia, and the initial minutes of reperfusion). For that, we used isolated perfused hearts from Lewis rats (female/male) and ischemic preconditioning (IPC: three cycles 5/5 min IR) as the most robust cardioprotective stimulus (infarct size reduction of ~68%; Physiol Rep. 2019 Jun 17;7(12):e14146). Hearts were subjected to 30 min global ischemia and 10 min reperfusion with/without preceding IPC. In subgroups the protocols were terminated before IPC, after IPC or the respective time control, at 25 min of ischemia and 10 min of reperfusion (n=10 hearts per group, respectively). Cardiac tissue samples were harvested and snap frozen and in parallel cardiac tissue was used to isolate mitochondria via differential centrifugation for mitochondrial respiration measurements. In cardiac tissue lysates, the mitochondrial fission (total and pS616 Drp1) and fusion (Mfn1/2 and Opa1) proteins were quantified via Western blotting and normalized to the mitochondrial housekeeper voltage-dependent anion channel expression. IPC induced rapid modification of Drp1 phosphorylation at pS616 whereby total Drp1 remains unchanged. In comparison to the time control group without IPC, Drp1 phosphorylation was increased (1.35±0.22 vs. 0.95±0.19) directly after IPC, but lesser during ischemia (1.06±0.24 vs. 1.42±0.37), and not different phosphorylated at 10 min reperfusion (1.30±0.31 vs. 1.37±0.32). We confirm proteolytic cleavage of L-OPA1 to S-Opa1 by ischemic stress. However, S-Opa1 level were further increased by IPC (1.3±0.16 vs. 1.11±0.14) during ischemia, whereas L-Opa1 decreased (0.58±0.17 vs. 0.73±0.15). The other fusion proteins Mfn1/2 remained unchanged. Interestingly there were also rapid modifications of mitochondrial respiration by IPC, as directly after the maneuver ADP-stimulated respiration was increased in comparison to the group without IPC (236±24 vs. 146±15 nmol O 2 /min/mg protein). In conclusion, we here characterized for the first time the rapid regulation of mitochondrial fission and fusion mediators by myocardial IR injury and IPC. Future approaches have to identify, whether Drp1 and Opa1 play a causal role in respiration regulation by IPC and whether mitochondrial dynamics are also subject to such rapid changes. Funding: C.E. and P.K. acknowledge the support by the DFG (RTG 2989 P5), P.K. by the EU Cost Action METAHEART (CA22169), C.E. by the DGK (DGK05/2025), A.L. by the NIEHS (P30 GM103507) and Gheen’s Foundation, and A.B. by the NHLBI (R01HL173549). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.