Fig 2: Adipsin regulates ferroptosis pathway in vitro. (A) Flowchart of the study design. Neonatal mouse ventricular cardiomyocytes were treated with recombinant Adipsin protein. Following that, cardiomyocytes were exposed to hypoxia for 6–8 h. Next, RNA sequencing was performed on the cell genome. (B,C) Pathway enrichment investigation. (D) Under hypoxia, the Volcano graph shows differentially expressed genes. (E) The Heatmap of differentially expressed genes about ferroptosis. (F) Using quantitative PCR, the expression profile of ferroptosis-related genes in cardiac tissue post MI was investigated after Adipsin-enriched pericardial-AT exosomes treatment. n = 3, *p < 0.05 vs. MI + Vehicle group. Data were mean ± SEM, and one-way ANOVA was used for statistical analysis.

Fig 3: Irp2 is essential to Adipsin-offered cardioprotection. (A) Flowchart of the study design. Myocardial infarction surgery was performed 14 days after injection of AAV9 (Scramble or IRP2-shRNA) at the area of the myocardium. Following that, MI mice were injected four times with exosomes isolated from Adipsin-Tg or NTg mice’s pericardial adipose tissues. Cardiac function was assessed 28 days after MI surgery. Each group n = 6. (B,C) Representative echocardiographic images and left ventricular ejection fraction (LVEF) analysis. Each group n = 6. *p < 0.05 vs. AAV9-Scramble + Exo (NTg) group. (D) Non-heme iron levels were measured in the peri-infarct region of heart. Each group n = 6. *p < 0.05 vs. AAV9-Scramble + Exo (NTg) group. (E,F) Quantification of fibrotic area and representative images of Masson’s trichrome staining of the transverse planes post myocardial infarction. Each group n = 6. *p < 0.05 vs. AAV9-Scramble + Exo (NTg) group. (G) Malondialdehyde (MDA) levels in the peri-infarct region of heart. Each group n = 6. *p < 0.05 vs. AAV9-Scramble + Exo (NTg) group. (H–L) TFRC, FTH, COX2, and GPX4 proteins were measured by Western blots. Each group n = 6. *p < 0.05 vs. AAV9-Scramble + Exo (NTg) group. Data were mean ± SEM, and one-way ANOVA was used for statistical analysis.

Fig 4: Schematic diagram. Illustration of time-course of Adipsin levels following MI challenge and the role of exogenous Adipsin-evoked cardioprotection. A reduction of Adipsin expression (red line) is visible during the initial stage of MI. Exogenous administration of Adipsin via exosomes (blue dashed line) triggers cardioprotection, relieves cardiac remodeling, and improves cardiac function. MI, myocardial infarction; IRP2, Iron regulatory protein 2; ROS, reactive oxygen species.

Fig 5: Acute myocardial infarction evokes Adipsin production and secretion. (A) Serum Adipsin levels at admission and 1 month after discharge in MI patients. n = 16 and 14, *p < 0.05 vs. Control group. (B) Schematic illustration of animal experimental procedures analyzing Adipsin production and secretion. (C) Adipsin mRNA expression determined in pericardial adipose tissue (PAT), subcutaneous adipose tissue (SAT), brown adipose tissue (BAT) and other organs using RT-qPCR. n = 6, *p < 0.05 vs. Sham group (PAT), Ip < 0.05 vs. Sham group (SAT), §p < 0.05 vs. MI group (SAT). (D–G) Adipsin protein levels of pericardial adipose tissue (PAT), serum and heart tissues determined using Western blots. n = 6 (sham) and 42 (MI). *p < 0.05 vs. Sham group. For (A,C,E,F,G), statistical analysis was performed using one-way ANOVA.