IRE1a links lipid stress to a novel interactome in cardiac lipotoxicity

Abstract Rationale Obesity and metabolic dysfunction drive HFpEF, but the mechanism by which lipid overload drives disease progression remain unclear. IRE1α, a key UPR sensor, is dysregulated in HFpEF yet whether IRE1α functions as a cardiomyocyte lipid stress sensor and how its interactome is altered during cardiac lipotoxicity remain poorly understood. Objectives We hypothesized that IRE1α is a critical signaling hub for cardiomyocyte lipotoxicity and aim to 1) identify lipid species that selectively activate IRE1α, 2) define how saturated lipids modulate IRE1α signaling and 3) map its interactome under lipid stress 4) elucidate the structural biology behind the interaction. Methods We developed cardiomyocyte reporter cell lines to track IRE1α-XBP1s activation upon lipid treatment. Flow cytometry and biochemical assays were used to validate these models. Lipid screening identified unique lipid species that activate IRE1α. A TurboID-based proximity-labeling approach mapped IRE1α interactors, while FRET assays with YFP- and mCherry-tagged vectors validated protein interactions. Furthermore, molecular brightness experiment and computational modeling provided insights on the structural assembly of the interactome binding with IRE1α. Results The fatty acid palmitate, but not oleate, triggered strong IRE1α activation and XBP1s splicing, despite both inducing lipid accumulation in cardiomyocytes. Using the AC16-UPRG-mCherry reporter line, we confirmed that only palmitate activates IRE1α signaling. Medium-throughput lipid screening identified additional lipid species activating the IRE1α-XBP1s pathway. Proteomic analysis using the AC16-IRE1α-TurboID system revealed a direct interaction between IRE1α and MyD88, an inflammatory adaptor protein. Co-immunoprecipitation (Co-IP) further confirmed that MyD88 physically associates with IRE1α specifically in the presence of palmitate. FRET assays showed a significant increase in interaction between IRE1α and MyD88 upon palmitate treatment, which was not observed with MyD88 and PERK. Silencing MyD88 reduced XBP1s splicing and its downstream targets upon palmitate. Molecular brightness experiments revealed the structural assembly through which MyD88 interacts with IRE1α under lipid stress, and computational modeling identified the key IRE1α domains mediating this interaction. Conclusion Different lipid species differentially regulate IRE1α signaling. We identify MyD88 as a novel lipid-induced interactor, linking ER stress to inflammatory pathways in cardiomyocytes, offering new insights into HFpEF and cardiac lipotoxicity.For image description, please refer to the figure legend and surrounding text.