Epicardial adipose tissue browning attenuates cardiomyocyte ferroptosis in ischemia/reperfusion injury by suppressing ACSL1 cargo loading in M1 macrophage-derived small extracellular vesicles

Background Ferroptosis plays a central role in myocardial ischemia/reperfusion (MI/R) injury. M1 macrophage-derived small extracellular vesicles (M1-sEVs) deliver protein cargo that promote lipid peroxidation. Epicardial adipose tissue browning reduces local inflammatory response and remodels lipid metabolism, gowever, whether it modulates cardiomyocyte ferroptosis via the ACSL1 cargo loading in M1-sEVs remains unclear. Purpose To investigate how EAT browning attenuates cardiomyocyte ferroptosis and alleviates MI/R injury by suppressing M1 macrophage polarization and ACSL1 cargo loading in M1-sEVs. Methods Peripheral sEVs were collected from patients with acute coronary syndrome (ACS, n=150), chronic coronary syndrome (CCS, n=150) and healthy controls (HC, n=50) for sEVs extraction. ACSL1 expression in sEVs was measured by western blotting (WB) and ELISA, and correlated with cardiac injury markers. M1-sEVs were isolated from LPS-stimulated BMDMs and identified. LFQ proteomics was used to analyze differentially expressed proteins. Brown adipocyte markers and macrophage polarization markers were measured. Mouse MI/R and cardiomyocyte hypoxia/reoxygenation (H/R) models were constructed. Cardiac function/infarct size were assessed by echocardiography/TTC. Ferroptosis was assessed by lipid peroxidation, ROS, iron, MDA/GSH and GPX4. An ACSL1 knockdown model in M1 macrophages was established using siRNA and ferroptosis was inhibited by Fer-1. Results Elevated ACSL1 expression in sEVs was higher in ACS group than CCS and HC groups and positively correlated with cTnI, CK-MB and BNP. LFQ proteomics identified ACSL1 as an enriched ferroptosis-related cargo protein in M1-sEVs. In vitro, M1-sEVs were efficiently taken up by cardiomyocytes and exacerbated ferroptosis under H/R, as evidenced by decreased cell viability, elevated lipid peroxidation and oxidative stress, iron accumulation, and reduced GPX4 levels. These changes were significantly reversed by Fer-1. In vivo, the MI/R+ M1-sEVs group exhibited severe myocardial injury compared to the group, evidenced by worsened cardiac function, increased infarct size, reduced GPX4 levels, and elevated lipid peroxidation. EAT browning upregulated UCP1expression, reduced M1 polarization, and decreased ACSL1 cargo loading in M1-sEVs, accompanied by reduced ferroptosis-related signals, improved cardiac function, and reduced infarct size after MI/R. siACSL1 in M1 macrophages inhibited cardiomyocyte ferroptosis, protected the injured myocardium, and promoted cardiac function recovery. Conclusion EAT browning attenuates cardiomyocyte ferroptosis and MI/R injury by suppressing ACSL1 enrichment in M1-sEVs. ACSL1 appears to be a pivotal mediator of ferroptosis in MI/R injury, suggesting potential therapeutic approaches of promoting EAT browning and targeting ACSL1 cargo loading in M1-sEVs.