Hypoxia Microenvironment Preconditioning Attenuated Myocardial Ischemia‐Reperfusion Injury via Stc1‐Mediating Cardiomyocyte Self‐Protection and Neutrophil Polarization

Ischemic preconditioning (IPC) therapy application to attenuate myocardial ischemia-reperfusion (MI/R) injury in clinical practice remains challenging. The secretome, derived from hypoxia-preconditioned cardiomyocytes (SHPC), potentially mimics the IPC microenvironment and facilitates IPC clinical translation. This study aims to determine whether SHPC can be a feasible alternative to IPC for attenuating MI/R injury, and to identify the functional factor of SHPC. The ultrafiltration technique is applied to generate an SHPC formulation that is intramyocardially injected before reperfusion in a murine MI/R model. The effects of SHPC on cardiomyocyte apoptosis, pyroptosis, and neutrophil polarization are evaluated. Secretomics, neutralizing antibodies, and recombinant proteins are employed to identify the functional factor in SHPC. Co-immunoprecipitation assays, RNA sequencing, and site-directed mutagenesis are conducted to investigate the underlying mechanism. Additionally, a recombinant functional factor-encapsulated hydrogel is developed for intrapericardial injections (iPC). An intramyocardial SHPC injection in MI/R-injured mice strikingly reduces infarct size and the expression of cardiac injury biomarker while improving cardiac function. SHPC eliminated mitochondrial reactive oxygen species and triggered neutrophil polarization to reduce cardiomyocyte apoptosis/pyroptosis upon hypoxia/reoxygenation injury. Stanniocalcin 1 (Stc1) is identified as the functional factor in SHPC, mediating hypoxic microenvironment. Mechanistically, hypoxia-preconditioned cardiomyocytes secrete Stc1 into the microenvironment and activate calcium-sensing receptor (CaSR) that increases Stat3 phosphorylation at Ser727 via nitric oxide synthase 2 (NOS2)-mediated S-nitrosylation, thereby decreasing cardiomyocyte apoptosis/pyroptosis in an autocrine mechanism. Simultaneously, Stc1 facilitates cardiomyocyte-neutrophil crosstalk, thereby triggering neutrophil polarization to reduce inflammatory damage via the CaSR/NOS2/Stat3 axis in a paracrine mechanism. Pericardial delivery of a recombinant rStc1-encapsulated hydrogel has extended the therapeutic time window of rStc1, improving long-term cardiac function. The hypoxia microenvironment preconditioning, which mimicked by SHPC, attenuated MI/R injury via Stc1-mediated cardiomyocyte self-protection and neutrophil polarization. This study suggests that SHPC, with hypoxia preconditioning factor Stc1, represents a clinically feasible alternative to IPC for attenuating MI/R injury.