NUPR1-dependent metallothionein-2 transcription attenuates ferroptosis-associated myocardial injury in diabetic cardiomyopathy

Background Diabetic cardiomyopathy (DbCM) develops in a setting of sustained glucolipotoxic stress, mitochondrial dysfunction, and maladaptive myocardial remodeling. Ferroptosis-associated lipid peroxidation and iron dysregulation have been implicated in diabetic myocardial injury, but the stress-responsive transcriptional programs that restrain this process remain incompletely defined. We investigated whether nuclear protein 1 (NUPR1) protects the diabetic heart through transcriptional regulation of metallothionein-2 (MT2). Methods DbCM was induced in male C57BL/6J mice by high-fat feeding followed by streptozotocin. Cardiac NUPR1 was increased or reduced using adeno-associated virus serotype 9 vectors, and primary neonatal mouse cardiomyocytes were exposed to high glucose plus palmitate for complementary gain- and loss-of-function studies. Cardiac function, remodeling, mitochondrial ultrastructure, iron accumulation, lipid peroxidation, and ferroptosis-associated proteins were assessed. RNA sequencing, promoter-reporter assays, and chromatin immunoprecipitation were used to identify and validate NUPR1-regulated targets. LNP-formulated NUPR1 mRNA and zinc supplementation were evaluated as proof-of-concept interventions. Data were analyzed using two-tailed t tests, one-way ANOVA, or two-way ANOVA for factorial designs, with multiplicity-adjusted post hoc comparisons as appropriate. Results NUPR1 expression increased in diabetic hearts and glucolipotoxic cardiomyocytes. NUPR1 knockdown worsened cardiac dysfunction, hypertrophy, fibrosis, mitochondrial injury, iron accumulation, and lipid peroxidation, whereas NUPR1 overexpression produced the opposite effects. Ferrostatin-1 attenuated the vulnerability associated with NUPR1 loss, supporting a contribution of ferroptosis-associated injury. Transcriptomic analysis identified Mt2 as a metal-homeostasis candidate downstream of NUPR1. NUPR1 increased Mt2 promoter activity and occupied the Mt2 promoter in cardiomyocytes, while MT2 silencing weakened NUPR1-mediated cytoprotection in vitro. Systemic NUPR1 mRNA-LNP administration increased myocardial NUPR1 and MT2 expression and improved cardiac function and remodeling in DbCM mice. Zinc supplementation produced limited functional benefit in unselected DbCM mice but improved the aggravated phenotype associated with NUPR1 deficiency. Conclusions NUPR1 functions as an adaptive myocardial stress-response factor that limits iron-associated oxidative injury in DbCM, in part through transcriptional activation of MT2. The NUPR1-MT2 axis therefore represents a candidate cardioprotective pathway and a potential therapeutic entry point that warrants in vivo MT2 epistasis and human validation.