Hypoxia preconditioning enhances the cardioprotective effects of induced regulatory T cells after myocardial infarction through HIF-1α-mediated mechanisms.

Regulatory T cells (Tregs) are pivotal in resolving inflammation and promoting cardiac repair after myocardial infarction (MI). However, their therapeutic potential is constrained by cell availability, poor survival in ischemic myocardium, and inefficient homing to injured tissue. Hypoxia preconditioning (HP) is a priming strategy that mimics hypoxic stress and enhances cellular adaptation to hostile microenvironments. This study investigated whether HP could improve the therapeutic efficacy of iTregs. In a mouse MI model, we demonstrated that HP-iTregs significantly improved cardiac function, reduced inflammatory responses, enhanced angiogenesis, and attenuated myocardial fibrosis compared with normoxia-cultured iTregs. Transcriptomic profiling showed upregulation of hypoxia-inducible factor-1α (HIF-1α) and downstream genes associated with cell migration, tissue retention, and paracrine activity, validated by flow cytometry and ELISA. In vitro co-culture showed that conditioned medium derived from HP-iTregs promoted the viability and functional activity of cardiac microvascular endothelial cells and mitigated hypoxia-induced injury in cardiomyocytes. These protective effects were substantially diminished when conditioned medium was obtained from HIF-1α-deficient HP-iTregs. Furthermore, the beneficial effects of HP were markedly attenuated in iTregs generated from CD4+ T cell-specific HIF-1α knockout mice, supporting a central role for HIF-1α in mediating the enhanced cardioprotective properties of HP-iTregs. Immunofluorescence analysis of exogenously transferred EGFP+ iTregs further showed that HP increased the accumulation of iTregs in the infarcted myocardium, whereas this effect was attenuated in HIF-1α-deficient HP-iTregs. HP represents a practical ex vivo strategy to improve the therapeutic performance of iTregs following MI by activating an HIF-1α-mediated program that promotes their cardiac accumulation and paracrine function.