Exercise training alleviates myocardial injury in mice with heart failure with preserved ejection fraction via the TLR4-mediated necroptosis pathway.

Abstract

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a prevalent cardiovascular disorder with limited evidence-based therapies. Although high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) improve cardiac function in patients, the optimal exercise intensity and underlying molecular mechanisms remain poorly understood. We used an HFpEF mouse model to systematically assess how exercise training affects myocardial injury, specifically examining the TLR4-mediated necroptotic pathway. METHODS: To evaluate the effects of exercise training, mice were randomly assigned to four groups: Control, HFpEF, HFpEF+HIIT, and HFpEF+MICT. After 6 weeks of exercise, we performed echocardiography and histological staining to assess cardiac structure and function. Subsequently, we performed transcriptome sequencing to identify differentially expressed genes and key signaling pathways. These were validated by Western blot, quantitative PCR (qPCR), histological staining, and transmission electron microscopy. For mechanistic validation, small interfering RNA (siRNA)-mediated knockdown of TLR4 was used to clarify its regulatory role in necroptosis induced by "double-damage" challenge in H9C2 cardiomyocytes. RESULTS: HIIT and MICT both improve hypertension, impaired glucose tolerance, reduced exercise tolerance, and cardiac diastolic dysfunction in mice with HFpEF. They also attenuate cardiac hypertrophy and myocardial fibrosis. Notably, HIIT produces the most pronounced effects. Transcriptomic profiling revealed necroptosis to be the pivotal pathway mediating the protective effects of exercise. Concurrently, mitochondrial function was remodeled. TLR4 was identified as the upstream master regulator of this axis. In a "double-damage" H9C2 cardiomyocyte model, silencing TLR4 suppressed necroptosis-related genes and partially restored mitochondrial membrane potential. CONCLUSION: In HFpEF, the cardioprotective effects of exercise training, particularly HIIT, may be attributed to the inhibition of TLR4-mediated necroptosis. These findings indicate that therapeutic strategies targeting TLR4 and necroptosis may confer clinical benefit in HFpEF. Furthermore, exercise training constitutes an effective non-pharmacological intervention that modulates this pathway.