Aconitine alleviates myocardial hypertrophy and heart failure via targeting Septin4 ubiquitination

Background: Heart failure (HF) is the terminal stage of cardiovascular diseases, with currently limited therapeutic strategies available. The small GTPase Sept4 (Septin4) has been shown to mitigate myocardial remodeling and even improve HF. Aconitine (AC), derived from Fuzi, is a well-proved and frequently-used HF-targeting active natural compound. However, the exact underlying mechanisms by which Septin4 regulates HF and its interaction with AC remain elusive.Purpose: This study aims to elucidate the potential mechanisms by which AC suppresses myocardial hypertrophy and HF via Septin4-associated mechanisms.Methods: Isoproterenol (ISO)-induced myocardial hypertrophy and HF model was used to evaluate the cardioprotective effect of AC. Solvent-induced protein precipitation (SIP) combined with proteomic analysis was employed to gain the AC-binding proteins. CETSA and DARTS were further applied for identifying the binding of AC with Septin4. The co-immunoprecipitation assay, cell transfection, ubiquitination assay, and site-specific mutation were performed to detect the interaction between UCHL1 and Septin4, as well as the ubiquitination site where UCHL1 regulated Septin4 degradation.Results: AC alleviated ISO-triggered cardiac hypertrophy and HF both in vitro and in vivo models, via downregulating the HF biomarkers ANP and BNP and restraining cardiomyocyte apoptosis. Subsequently, Septin4 is identified as a potential target of AC with a high-affinity by SIP coupled with proteomic analysis. Crucially, AC physically interacted with the GTPase domain of Septin4, thereby promoting its degradation. Accordingly, AC reversed the effect of Septin4 overexpression and impeded its protective role when Septin4 was deleted, further confirming Septin4 as the bona fide target of AC. Mechanistically, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) bound to the GTPase domain of Septin4, ubiquitinating Septin4-K290 to degrade Septin4 via the ubiquitin-proteasome system, whereas AC further alleviated myocardial hypertrophy and HF by hindering the binding of Septin4 to UCHL1 and promoting Septin4 degradation.Conclusion: Our findings provide the first evidence that Septin4 serves as a novel biomarker for determining the cardioprotective effects of AC, offering a promising therapeutic approach for cardiac hypertrophy and HF.