Data-Independent Acquisition Proteomics and N-Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts

Abstract

Myocardial ischemia-reperfusion (IR) (stunning) injury triggers changes in the proteome and degradome of the heart. Here, we utilize quantitative proteomics and comprehensive degradomics to investigate the molecular mechanisms of IR injury in isolated rat hearts. The control group underwent aerobic perfusion, while the IR injury group underwent 20 min of ischemia and 30 min of reperfusion to induce a stunning injury. As MMP-2 activation has been shown to contribute to myocardial injury, hearts also underwent IR injury with ARP-100, an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2 to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy, we quantified 4468 proteins in ventricular extracts, whereby 447 proteins showed significant alterations among the three groups. We then used subtiligase-mediated N-terminomic labeling to identify more than a hundred specific cleavage sites. Among these protease substrates, 15 were identified following IR injury. We identified alterations in numerous proteins involved in mitochondrial function and metabolism following IR injury. Our findings provide valuable insights into the biochemical mechanisms of myocardial IR injury, suggesting alterations in reactive oxygen/nitrogen species handling and generation, fatty acid metabolism, mitochondrial function and metabolism, and cardiomyocyte contraction.

N-Terminomic Identification of Intracellular MMP-2 Substrates in Cardiac Tissue

“…To assess the biological implications of the identified substrates, we compared our data to the literature on hypoxia-reoxygenation or ischemia-reperfusion injury in the heart or cardiomyocytes. − We then compared substrates identified here to those cleaved by MMP-2 in vitro. − In addition, we took into account our accompanying paper, describing the degradome changes that occur during cardiac ischemia-reperfusion injury in rat hearts (Table S2, Supporting Information). By comparing the results between these studies, we identified multiple proteins that were cleaved at the same site following ischemia-reperfusion injury and by MMP-2-Fc-induced proteolysis in ventricular extracts.…”

History of the development of isolated heart perfusion experimental model and its pioneering role in understanding heart physiology

“…High-resolution imaging techniques like MRI and PET are used to provide real-time, detailed images of cardiac function, while integration with bioengineering has led to the development of 3D-printed heart tissues and organs-on-chips that simulate more realistic cardiac physiology [ 50 – 52 ]. Additionally, advanced genomic and proteomic techniques allow for detailed analysis of changes within the heart under various conditions, aiding in the discovery of new therapeutic targets [ 53 ]. Moreover, the isolated perfusion model expands opportunities for targeted immunomodulation, building tolerance to ischemia-reperfusion injury, and correcting pathogenic genetic defects.…”