Updated project metadata. Advancements in cross-linking mass spectrometry (XL-MS) for structural analysis of proteins bridge the gap between purified systems and native tissue environments. Here, isobaric quantitative protein interaction reporter technology (iqPIR) was utilized to further extend XL-MS to the first system-wide comparative study of mitochondrial proteins from healthy and diseased murine hearts. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition. Structural insight into ketone-oxidation metabolons, OXPHOS machinery, and nucleotide transporter hybrid-conformations support mitochondrial remodeling in failing hearts while bringing forth new hypotheses for pathological mechanisms. The application of quantitative cross-linking technology in tissue provides molecular-level insight into complex biological systems challenging to model in cell culture, thus providing a valuable resource for studying human diseases.