Background: Isoproterenol (ISO) induces oxidative and apoptotic damage in cardiomyocytes. This study aimed to investigate whether hesperidin (HES) confers a protective effect against ISO-induced injury in H9c2 cardiomyocytes and to determine whether this effect modulates the Protein Kinase B (Akt)/Glycogen Synthase Kinase-3 Beta (GSK3β) signaling pathway.

Methods: H9c2 rat embryonic cardiomyocytes were treated with ISO to establish the optimal injury concentration and with varying HES doses (5, 10, 20 μM) to define a safe and effective range (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT assay). The cells were divided into five groups: Control, ISO alone, and ISO plus HES at each concentration. The viability and proliferative capacity of the cells were assessed using MTT and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Markers of injury and oxidative stress—Lactate dehydrogenase (LDH) release, glutathione (GSH), catalase (CAT), and malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and intracellular reactive oxygen species (ROS)—were measured. Apoptosis and mitochondrial function were assessed using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC‑1) dye for membrane potential, and Western blotting for B-cell lymphoma 2 (Bcl‑2), Bcl-2-associated X protein (Bax), Cysteine-aspartic acid protease-3 (caspase‑3), PTEN-induced putative kinase 1 (PINK1), Parkin and cytochrome C. Akt/GSK3β pathway activation was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and phospho‑specific Western blotting. The role of Akt was confirmed by co-treatment with the Akt-specific inhibitor VIII.

Results: Compared to ISO alone, HES dose-dependently enhanced cell viability and proliferation, significantly reduced LDH release, MDA, and ROS levels, while increasing GSH and CAT levels, as well as SOD activity (p < 0.05). Results from the TUNEL assay and JC-1 staining demonstrated that treatment with HES markedly reduced the proportion of apoptotic cells and effectively maintained the integrity of the mitochondrial membrane potential (p < 0.05). Western blot analysis showed that HES treatment upregulated the levels of anti-apoptotic protein Bcl-2 and mitochondrial quality control proteins PINK1 and Parkin, while downregulating the levels of pro-apoptotic proteins Bax, caspase-3, and cytochrome C (p < 0.05). Treatment with HES notably enhanced the phosphorylation levels of both Akt and GSK3β proteins (p < 0.05), indicating activation of this signaling cascade. These protective effects were abolished in the presence of Akt inhibitor VIII, confirming reliance on Akt/GSK3β signaling.

Conclusion: Hesperidin exerts significant cardioprotective effects by alleviating ISO-induced oxidative stress, mitochondrial impairment, and apoptotic injury in H9c2 cardiomyocytes, an effect largely attributed to its activation of the Akt/GSK3β signaling pathway, thereby highlighting its therapeutic potential in cardiac protection.