〔Abstract〕 Objective To examine the impact of Toll-like receptor 4 (TLR4)-mediated neutrophil extracellular traps (NETs) formation on isoproterenol (ISO)-induced heart failure (HF) in aged mice. Methods The mice were randomly divided into Control group, Model group, TAK-242 (TLR4 inhibitor) group, LPS (TLR4 agonist) group and LPS+DNaseⅠ (NETs inhibitor) group, with 8 mice in each group. The HF model was established by continuous infusion of ISO through an osmotic pump, and TAK-242, LPS, and LPS+DNaseⅠ interventions were administered during the last week of infusion for 7 consecutive days. ELISA was used to detect the N-terminal pro-B-type natriuretic peptide (NT-pro BNP), cardiac troponin I (cTnI), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and serum and myocardial tissue myeloperoxidase-DNA (MPO-DNA) and neutrophil elastase-DNA (NE-DNA) levels. HE staining was used to observe the pathological changes of myocardial tissue. Masson staining was used to observe the deposition of collagen fibers in myocardial tissue. Immunofluorescence was used to observe the colocalization of Ly6G and MPO in myocardial tissue. The protein expressions of TLR4, MPO, NE and cit-H3 in myocardial tissue were detected by Western blot. Results Compared with the Control group, the end-diastolic interventricular septal thickness (IVSD), left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) in the Mode group significantly increased ( P<0.01), fractional shortening (FS) and ejection fraction (EF) significantly decreased ( P<0.01), and the myocardial structure was significantly damaged. The myocardial collagen fiber precipitation, serum NT-pro BNP, cTnI, IL-1β, IL-6 and TNF-α contents, MPO-DNA and NE-DNA contents in serum and myocardial tissue significantly increased ( P<0.01). The co-localization of Ly6G and MPO and the expression of TLR4, MPO, NE and cit-H3 proteins in myocardial tissue significantly increased ( P<0.01). After TAK-242 intervention, the above indicators were significantly reversed, while the injury was further aggravated in LPS group. DNaseⅠ treatment could partially alleviate the deterioration of myocardial structure and function induced by LPS. Conclusion TLR4 and NETs are abnormally elevated in the myocardial tissue of aged HF mice. Treatment with the TLR4 inhibitor TAK-242 effectively suppresses NET formation, attenuates inflammatory responses, and enhances cardiac function in aged HF mice.