Effects of Heat Stress on the Fat Greenling ( Hexagrammos otakii ) Necroptosis Mediated by MLKL and Heart Injury

Mixed lineage kinase domain‐like pseudokinase (MLKL) is a key effector of necroptosis. We identified an MLKL homologue from Hexagrammos otakii , named Ho MLKL. The open reading frame (ORF) of Ho MLKL contained 1497 nucleotides and encoded a protein with 498 amino acids (Mw = 57.15 kDa). Bioinformatic analysis revealed that Ho MLKL is highly conserved. In the experiments, fish were exposed to 16°C (control), 24°C, and 28°C for up to 24 h, and samples were taken at 0, 6, 12, and 24 h. We used quantitative real‐time polymerase chain reaction (qRT‐PCR), Western blot, and immunohistochemistry (IHC) to quantify Ho MLKL and related protein in the cardiac tissue of H. otakii . Cardiac injury was evaluated by histopathology and biochemical assays. The results showed that Ho MLKL may play an important role in heat‐stress‐induced cardiac injury in H. otakii . Heat stress significantly upregulated Ho MLKL, receptor‐interacting protein kinase 1 (RIPK1), and receptor‐interacting protein kinase 3 (RIPK3) expression ( p < 0.05), indicating activation of the necroptosis pathway. Cardiac damage was characterized by elevated creatine kinase (CK) and lactate dehydrogenase (LDH), myocardial disorganization, vascular congestion, chromatin condensation, and inflammatory cell infiltration. Increased expression of inflammatory factors and reduced glucose levels suggested concomitant inflammation and metabolic dysfunction. These findings demonstrate that Ho MLKL likely cooperates with RIPK1/RIPK3 to mediate necroptosis, linking heat stress to cardiac injury and inflammation in H. otakii , and provide insight into stress‐induced necroptosis in fish.