Targeting the Toll-Like Receptor 4 Ameliorates Heart Failure in Aged Mice by Inhibiting the Formation of Neutrophil Extracellular Traps.

Heart failure (HF) is a prevalent cardiovascular condition among the elderly population, with an incidence rate that continues to rise annually, highlighting the urgent need for effective therapeutic interventions. Sustained activation of Toll-like receptor 4 (TLR4) may contribute to left ventricular dysfunction and adverse cardiac remodeling through the induction of myocardial inflammation and oxidative stress - pathological processes that closely align with the hallmark features of HF. Preclinical studies in animal models have demonstrated that TLR4 deficiency improves cardiac function in aged mice; however, the precise role and underlying mechanisms of TLR4 in human HF remain poorly understood. This study aims to test the central hypothesis that TLR4 serves as a critical molecular link between chronic inflammation and the pathophysiology of HF. HF was induced in 18-month-old male C57BL/6J mice via continuous subcutaneous infusion of isoproterenol (ISO, 30 mg/kg/day) over a period of 3 weeks. Thereafter, mice received daily intraperitoneal injections of the TLR4 inhibitor TAK-242 (2 mg/kg), deoxyribonuclease I (DNase I, 5 mg/kg), or the peptidylarginine deiminase 4 (PAD4) inhibitor GSK484 (4 mg/kg) for 7 consecutive days. Cardiac function was assessed using a ultrasound imaging system. HE staining and Masson staining were employed to evaluate myocardial pathological changes and collagen deposition. ELISA was performed to measure serum levels of myeloperoxidase-DNA (MPO-DNA), neutrophil elastase-DNA (NE-DNA), cTnI, NT-proBNP, IL-1beta, IL-6 and TNF-alpha. Immunofluorescence staining was performed to detect the co-localization levels of Ly6G with myeloperoxidase (MPO) and citrullinated histone H3 (cit-H3) in myocardial tissue, in order to assess the formation level of neutrophil extracellular traps (NETs). Western blot were utilized to determine the expression level of TLR4 protein. The expression of TLR4 was significantly upregulated in the myocardial tissue of aged HF mice. Inhibition of TLR4 not only markedly improved cardiac function but also alleviated pathological damage to myocardial tissue and reduced collagen fiber deposition. Concurrently, it also decreased the serum levels of MPO-DNA, NE-DNA, NT-proBNP, cTnI, and inflammatory factors. Moreover, the colocalization levels of Ly6G with MPO or cit-H3 in myocardial tissue was also diminished. These findings were consistent with the effects observed following DNase I and GSK484 interventions. Targeting TLR4 can mitigate inflammatory responses and enhance cardiac function in HF mice by inhibiting NETs formation. Key words Heart failure " Cardiac function " Inflammation " Toll-like receptor 4 " Neutrophil extracellular traps.