Septin4 mediates hydrogen peroxide-induced cardiomyocyte damage by initiating PARP1-dependent apoptosis

Objectives: Although Septin4 has been shown to play a role in apoptosis regulation across various cell types, its specific function and mechanism in oxidative stress-mediated cardiomyocyte apoptosis remain unclear. This study aimed to investigate the role of Septin4 and its underlying molecular mechanism in hydrogen peroxide-induced cardiomyocyte apoptosis. Methods: To model myocardial ischemia-reperfusion injury, H9C2 cells and primary rat cardiomyocytes were treated with hydrogen peroxide to induce oxidative stress. Cell apoptosis was assessed using cell counting kit-8 assay, flow cytometry, and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. Protein expression levels of Septin4, poly(ADP-ribose) polymerase 1 (PARP1), cleaved PARP1, Caspase3, cleaved Caspase3, PARylation, glyceraldehyde-3-phosphate dehydrogenase, 3-nitrotyrosine, 8-oxoguanine-DNA glycosylase, and superoxide dismutase 1 were analyzed by western blotting. The interactions between cleaved Caspase3 and Septin4, PARP1 and Septin4, and cleaved Caspase3 and PARP1 were examined by coimmunoprecipitation. Functional effects and loss-of-function studies of Septin4 were performed using plasmid overexpression and shRNA/siRNA knockdown. Results: Septin4 expression in cardiomyocytes increased in a dose- and time-dependent manner following hydrogen peroxide treatment. Overexpression of Septin4 exacerbated cardiomyocyte apoptosis, while knockdown of Septin4 attenuated these effects. Mechanistically, Septin4 physically interacted with both cleaved Caspase3 and PARP1, forming a ternary complex that enhanced the cleavage of PARP1 by cleaved Caspase3 in cardiomyocytes. Conclusions: Septin4 promotes oxidative stress-induced cardiomyocyte apoptosis by facilitating the cleavage of PARP1 by cleaved Caspase3, acting as a molecular bridge within the cleaved Caspase3-Septin4-PARP1 axis. This study identifies Septin4 as a novel modulator of cardiomyocyte apoptosis and suggests it as a potential therapeutic target for mitigating myocardial ischemia-reperfusion injury.