Heat stress promotes ferroptosis in Jiaji duck myocardium by disrupting iron homeostasis and inducing lipid peroxidation.

Heat stress is a common environmental challenge in poultry production, causing substantial economic losses and acute mortality. The heart is highly sensitive to thermal load and is a major target of heat-induced injury. Although heat stress-related myocardial injury in poultry has been linked to oxidative stress and apoptosis, the contribution of ferroptosis remains unclear. Here, we used the Jiaji duck as a model to investigate the molecular mechanisms of heat stress-induced myocardial injury. We established an acute heat stress model in Jiaji ducks (40 ± 1°C, 80% relative humidity, 3 h) and an in vitro heat stress model in primary cardiomyocytes, and performed a multi-platform analysis. Acute heat stress reduced survival in a time-dependent manner, increased serum CK-MB and LDH levels, and induced myocardial lesions characterized by myofiber disorganization and disruption. Transmission electron microscopy revealed mitochondrial shrinkage and loss of cristae. Whole-proteome profiling identified 1,011 differentially expressed proteins, with enrichment of ferroptosis-related terms and pathways and concurrent enrichment of AMPK signaling. Biochemical analyses showed increased ROS and MDA levels and elevated ferrous iron (Fe²⁺), accompanied by glutathione (GSH) depletion and reduced GPX4 abundance. Consistently, western blotting indicated activation of the AMPK/ULK1 axis (increased p-AMPK and p-ULK1), upregulation of TFR1 and ACSL4, and downregulation of FTH1 and SLC7A11. In vitro, heat stress elicited similar molecular changes together with increased CK-MB/LDH release and reduced viability, supporting a direct cardiomyocyte response. Collectively, these findings indicate that heat stress promotes a ferroptosis-like injury program in Jiaji duck cardiomyocytes, characterized by iron accumulation, impaired GSH-GPX4 antioxidant defense, and enhanced lipid peroxidation, and is associated with activation of AMPK/ULK1 signaling. These results provide mechanistic insights and potential targets for mitigating heat stress-induced myocardial injury in poultry.