Abstract Body (Do not enter title and authors here): Introduction: Prior research in our lab has identified that endothelial cells (EC) genetically modified to overexpress SOD2 (SOD2-OE) present mitochondria with higher oxidative phosphorylation (OXPHOS) and respiratory capacity, resulting in improved cardiac function in vivo in an acute myocardial infarction (MI) model.
Research question: We sought to elucidate the mechanisms involved in the higher OXPHOS in SOD2-OE ECs. For that, mouse proteome and human MI multi-omic datasets were analyzed.
Methods: We analyzed the proteome of SOD2-OE mouse heart EC (MHEC) for upregulated proteins. To understand how SOD2 mediates its effect on OXPHOS, we generated an interaction map on STRING, filtering proteins by subcellular localization and transcription regulation activity. Next, with the identified proteins, we analyzed the human spatial multi-omic map of MI to verify translational potential. We then performed siRNA knockdown of selected proteins in MHEC, investigating their effects on protein expression by western blot.
Results: Of the 68 upregulated proteins in the mouse proteome (SOD2-OE ECs vs. normal expression control ECs), we found 11 proteins upregulated in mitochondria and 27 upregulated in nucleus. Filtering for mitochondria and nucleus with transcriptional regulation activity, 22 proteins were identified. Of these, only one transcription regulator, Arl2bp, was present in nucleus, cytoplasm, and mitochondria (with 3.9 abundance ratio in SOD2-OE ECs versus the control, p<0.0001). Arl2bp is the binding pair of Arl2, which interacts with ANT1 (responsible for ADP/ATP shuttling). A second protein mrps28 (100 abundance ratio in SOD2-OE ECs vs. the control, p<0.0001) was identified to have a high confidence functional interaction with SOD2 (0.872) and GPx4 (0.815). Arl2bp and mrps28 were found decreased in human cardiac ECs after MI (by 44%, adj. p<0.0001, and by 28%, adj. p<0.0001, respectively), along with SOD2 (by 29%, adj. p<0.0001), GPx4 (by 38%, adj. p), and catalase (by 28%, adj. p<0.0001). In SOD2-OE MHECs, GPx4 knockdown decreased ANT1/2 expression (by 21%, p<0.05), while MRPS35 knockdown increased Arl2bp expression (by 82%, p<0.05).
Conclusion: We identified Arl2bp and mrps35 as two proteins that may contribute to higher OXPHOS induced by SOD2-OE in ECs. The role of these proteins in EC is unknown and the molecular mechanisms by which they modulate angiogenesis is being studied and will be presented at the conference.