Introduction Oxidative stress contributes to the functional decline of the myocardium and is correlated with unfavourable metabolic perturbations, including the switch from oxidative metabolism to glycolysis, branched chain amino acids and ketone bodies for energy production. Oxidative post-translational modifications (oxPTMs) might play a significant role in this process as they can influence both structure as well as the function of proteins. Methods We already demonstrated that end-stage failing human hearts endure higher levels of oxidative stress and exhibit higher oxidation of several different enzymes, namely related to glucose metabolism. We next aimed to unravel which oxidative changes might be connected with the early onset of oxidative damage. To that end, we simulated reperfusion injury (I/R) using cultured stem-cell derived and metabolically matured cardioids, followed by “one-pot” redox proteomics and small thiol analysis. The findings from cardioids were further corroborated in mouse model of myocardial infarction (MI), where the samples were carefully collected either thirty minutes or a week after the procedure from MI affected area (MI) of the left ventricle (LV); or remote, non-infarcted area of the same LV (R) or sham control (N=5 per condition/group). Results As a result, we report protein targets of acute oxidative damage - overlap of significantly redox affected cysteine residues between MI vs. R, MI vs. Sham and I/R vs. Control cardioids - many of which metabolic enzymes (Figure 1). In addition, we describe early perturbations of (metabolic) proteome in the infarcted area compared to its surroundings (and/or sham control), including downregulation of (oxidative) metabolic enzymes, and the rise of, among others, branched chain amino acid transaminase (Bcat1) as well as lysosomal acid lipase (Lipa). Lastly, we provide a unique insight into glutathione metabolism (including its precursors and intermediates) of both cardioids upon simulated I/R injury as well as LV tissue affected by MI, experimentally demonstrating the attempts of oxidation affected cardiomyocytes and the cardiac tissue to restore its glutathione supply. Conclusion Collectively our data provides novel insight into cardiac injury and suggest a potential direct cross-talk between redox signaling and metabolism upon oxidative stress exposure.