Abstract Body: Mitochondrial dysfunction is a main driver of decreased resilience and increased disease risk in the aging heart. Despite this, the specific structural remodeling of protein networks that drives bioenergetic deficits remains poorly understood. The goal of this study was to determine how structural changes in the cardiac mitochondrial complex I protein network influence cardiac function and mitochondrial bioenergetics during natural aging. We assessed the frailty index and cardiac function in young (8-mo; n=10) and aged (26-mo; n=8-10) NIA C57BL/6J mice of both sexes. Mitochondria isolated from young and old hearts were differentially labeled with reporter-heavy or stump-heavy Isobaric Quantitative Protein Interaction Reporter to quantify differences in cross-linked peptides (XLs) using mass spec. Changes in XLs were identified with a false discovery rate below 1%. We integrated quantitative cross-linking mass spectrometry with bioenergetics phenotyping (respiration and membrane potential) and complex I (CI) enzyme activity in isolated cardiac mitochondria. Aged mice exhibited a significantly elevated frailty index and impaired cardiac function, independent of sex. Mitochondrial interactome analysis uncovered a selective CI disassembly, particularly localized to the N and Q modules. Multiple interactions between the N and Q modules (e.g., NDUS4-NDUA6 and NDUA9-NDUS1 or NDUS6) were significantly reduced in the aged heart, suggesting structural loosening at the critical junction where these modules interface. Notably, NDUS4-relevant XLs were markedly reduced in aged hearts in both sexes, suggesting a selective vulnerability of this critical subunit with age. Conversely, subunit interactions within each module (e.g, N-N or Q-Q interactions) were not changed or increased. Complex I enzyme activity was also significantly impaired in males (p < 0.05), but not in females (p = 0.07). Complex I (glutamate- and pyruvate-malate)-fueled respiratory conductance was lower (p ≤ 0.05), and membrane potential was less polarized in aged hearts of both sexes. Our results demonstrate that aging-induced complex I disassembly contributes to bioenergetic failure and cardiac dysfunction, identifying the Complex I N-Q module as a potential molecular target to preserve mitochondrial integrity and treat age-related cardiovascular diseases.