Mutations in the Txnip PPXY motifs protect against myocardial infarction despite enhanced protein stability

Thioredoxin-interacting protein (Txnip), a member of α-arrestin superfamily, is best known for inhibiting thioredoxins and glucose transporters, increasing oxidative and metabolic stress. Through these functions, Txnip has emerged as a key contributor to the pathogenesis of heart diseases. Txnip contains C-terminal PPXY motifs that are conserved among α-arrestins across diverse species. Nevertheless, the physiological significance of these motifs remains unknown. We demonstrate that mutation of Txnip PPXY motifs to AAXA uncouples Txnip's canonical functions from its cytotoxic effects. Although the mutant reduced glutathione levels and glucose uptake to the same extent as wild-type Txnip, it attenuated cell death. To assess translational relevance, we engineered inducible, cardiomyocyte-restricted Txnip PPXY-to-AAXA knock-in mice. These mice displayed normal cardiac structure and function at baseline but were strongly protected after myocardial infarction, exhibiting improved left ventricular performance and overall survival. Mechanistically, structural modeling identified the E3 ubiquitin ligase ITCH as the principal PPXY-binding partner. The PPXY mutation abolished Txnip ubiquitination and stabilized Txnip protein, revealing a paradoxical dissociation between Txnip levels and cardiotoxicity. Transcriptomic profiling uncovered enrichment of PPAR-α/PGC-1α-associated pathways, although metabolic assays and untargeted lipidomics did not support these findings. Instead, immediate early genes of the Fos/Jun AP-1 network were consistently downregulated after ischemic injury, and AP-1 DNA-binding activity was diminished in PPXY mutant hearts. These results identify the Txnip PPXY motifs as key regulators of Txnip protein turnover and injury-responsive transcriptional programs. Disruption of these motifs stabilizes Txnip yet confers cardioprotection, providing evidence that elevated Txnip levels do not invariably drive cardiotoxicity.NEW & NOTEWORTHY This study reveals that Txnip's conserved PPXY motifs regulate its cytotoxicity independently of its canonical redox and metabolic functions. Mutating motifs (PPXY-to-AAXA) uncouples Txnip function from cell death, stabilizes the protein by preventing Itch-mediated ubiquitination, and confers striking cardioprotection in knock-in mice after myocardial infarction. Despite elevated Txnip levels, mutant hearts show reduced AP-1 signaling and improved survival, demonstrating that PPXY-dependent pathways, not Txnip abundance, drive cardiotoxicity.