Unveiling a novel pathway: Ginseng vanillic acid fights heart failure by coupling mitophagy to pyroptosis through the PINK1-BRCA1-NLRP3 axis

Abstract

Background

Heart failure (HF) represents a frequent and serious complication of hypertensive diseases. Substantial evidence has implicated angiotensin II (Ang II) in the promotion of cardiac remodeling and inflammation associated with hypertension. Vanillic acid (VA), an organic acid derived from natural sources such as ginseng, demonstrates a spectrum of bioactivities including anti-inflammatory and antioxidant effects.

Purpose

This study aimed to elucidate the potential of VA in attenuating Ang II-induced HF and to delineate its underlying mechanism of action.

Methods

In C57BL/6 mice, HF was induced by 4-week Ang II infusion. To delineate the cardioprotective mechanisms of VA, mice were treated with VA orally for 2 weeks following the initial 2 weeks of Ang II exposure, with subsequent cardiac RNA sequencing. VA-binding proteins were validated by molecular docking, DARTS, CETSA, and SPR.

Results

VA significantly ameliorated cardiac dysfunction without affecting blood pressure, concurrently suppressing cardiac fibrosis, hypertrophy, and inflammation. RNA sequencing implicated the PINK1 pathway, which was supported by in vitro observations of improved mitochondrial morphology and autophagic flux in NRVMs. Furthermore, direct binding between VA and BRCA1 was confirmed by molecular docking, DARTS, CETSA, and SPR.

Conclusions

This study demonstrates that VA suppresses NLRP3 inflammasome-mediated heart failure by restoring cardiomyocyte mitochondrial function through the PINK1- BRCA1 signaling axis. These findings identify VA as a promising therapeutic candidate for HF and support its further development as a novel agent targeting mitophagy and metabolic recovery in the stressed heart.

Graphical abstract

VA counteracts Ang II-induced heart failure by inhibiting cardiomyocyte pyroptosis through the activation of the PINK1-mediated mitophagy pathway and BRCA1.

1. Introduction

Heart failure (HF) represents a major global health challenge, affecting approximately 40 million people worldwide and accounting for substantial morbidity and mortality. Cardiac remodeling, which involves myocardial hypertrophy and fibrosis, is a fundamental process that underlies ventricular dysfunction in HF [1]. Among the diverse etiologies of HF, hypertension stands as a predominant risk factor. Hypertension-induced chronic pressure overload promotes myocardial hypertrophy and excessive extracellular matrix production, ultimately leading to impaired cardiac compliance and the clinical onset of HF [2].

During pathological cardiac remodeling, systemic and local activation of the renin-angiotensin system (RAS) leads to Ang II release. Initially compensatory, RAS activation helps maintain hemodynamic stability, with Ang II promoting adaptive responses such as physiological hypertrophy and angiogenesis [3]. However, sustained exposure to Ang II under pathological conditions drives a shift from adaptive responses to detrimental cellular processes [4]. Studies indicate that Ang II can induce excessive production of reactive oxygen species (ROS) by activating NADPH oxidase, triggering an oxidative stress cascade that subsequently impairs mitochondrial function in cardiomyocytes [5]. Notably, Ang II-induced mitochondrial dysfunction plays a pivotal role in the progression of HF, characterized by specific alterations including decreased mitochondrial membrane potential, impaired ATP synthesis, and abnormal mitochondrial dynamics [6]. Furthermore, Ang II can also induce pyroptosis [7]. Although angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) are the RAS inhibitors used in clinical practice, their application is limited by adverse effects including angioedema, hyperkalemia, and renal impairment [8,9]. These side effects not only compromise patient medication adherence but also restrict their widespread clinical application. Therefore, identifying novel therapeutic agents that can mitigate Ang II-induced cardiotoxicity with improved safety profiles is of great importance. Natural products have emerged as promising candidates for this purpose due to their multi-targeted actions and favorable biocompatibility.

Derived from natural sources including plants, animals, and minerals, botanical drugs are characterized by their complex chemical compositions, low tendency to induce microbial resistance, minimal toxicity. These properties endow them with significant potential in the prevention and treatment of HF. Vanillic acid (VA; 4-hydroxy-3-methoxybenzoic acid, Fig. 1B), a cinnamic acid derivative primarily isolated from Panax ginseng, is widely distributed in nature and abundantly present in various Chinese herbal medicines [10]. Extensive studies have demonstrated that vanillic acid exhibits diverse pharmacological activities, such as antihypertensive, antioxidant, anti-inflammatory, antifilarial, antibacterial effects, and snake venom neutralizing activity [11]. Across diverse disease models, VA demonstrates consistent therapeutic efficacy. In a collagen-induced arthritis, it alleviated symptoms and bone destruction, mitigated synovial and splenic pathology, and suppressed M1 macrophage polarization alongside pro-inflammatory mediator expression [12]. Similarly, VA exhibited protective effects in a model of liver fibrosis, VA promoted the MIF/CD74 signaling pathway in hematopoietic stem cell mitophagy, which may play a significant role in the pathogenesis of hepatic fibrosis [13], lung cancer [14], muscle atrophy [15], diabetic nephropathy [16] and chronic lymphocytic leukemia [17]. The underlying mechanisms involve the modulation of key pathways related to oxidative stress, inflammation, mitochondrial function, and cell death. Although VA demonstrates considerable therapeutic potential across numerous diseases, its specific role and mechanisms in cardiac disorders remain largely unexplored.

Fig. 1.

VA alleviates Ang II-induced cardiac dysfunction and cardiac remodeling in mice

Mice were treated as described in Methods. (A) Animal experimental procedures. (B) Chemical structure of VA. (C) Periodic systolic blood pressure measurement as determined by non-invasive tail-cuff pressure analysis system weekly. (D) Ang II levels in mouse serum were determined by ELISA kit. (E) Cardiac function in mice was determined by noninvasive echocardiography. (F–J) EF and ES of mice measured by ultrasonic scanning image system. (H)The ratio of heart weight to body weight was calculated. (I) Comparison of IRVT. (J) CK-MB levels in mice serum was determined by ELISA kit. All quantitative data are presented as mean ± SEM; n = 6; ns = not significant; **p < 0.01 versus the CON group; #p < 0.05, ##p < 0.01 versus the Ang II group, two-tailed Student's t-test.

This study systematically evaluated the therapeutic potential of VA against hypertensive HF using both in vivo (C57BL/6 mouse) and in vitro NRVMs models. By integrating RNA sequencing, TEM, Molecular docking, DARTS, CETSA, SPR techniques, flow cytometry and mitochondrial membrane potential assessment, we elucidated the cardioprotective mechanisms of VA, with a focus on its role in mitophagy and suppressing pyroptosis in hypertensive HF.

2. Materials and methods

Detailed methods are described in the Supplementary file.

3. Results

3.1. VA alleviates Ang II-induced cardiac dysfunction and cardiac remodeling in mice

We first evaluated whether VA exerts a protective effect against Ang II-induced cardiac injury in mice. A cardiac injury model was established by continuous infusion of Ang II (1000 ng/kg/min) via an osmotic minipump for 4 weeks. Starting from the 2 nd week after Ang II infusion, mice were treated with VA at doses of 25 mg/kg and 50 mg/kg via intragastric gavage, respectively (Fig. 1A). As shown in Fig. 1C and Fig. S1, Ang II infusion significantly increased blood pressure in mice, while VA treatment did not significantly reduce systolic blood pressure, indicating that VA lacks antihypertensive activity. Meanwhile, Ang II infusion led to a significant elevation in serum Ang II levels, which was not notably decreased in the VA treatment groups (Fig. 1D). These results suggest that VA does not exert cardiac protection by lowering blood pressure. However, assessment of cardiac function via echocardiography revealed that VA significantly alleviated Ang II-induced cardiac dysfunction in a dose-dependent manner (Fig. 1E)., as evidenced by improved EF and FS (Fig. 1F and G). Based on measurements of the HW/BW and isovolumic relaxation time (IVRT) (Fig. 1H and I), which was further confirmed by the quantitative data of HW/TL and a series of other cardiac parameters (Table S4). our data demonstrate that VA treatment alleviates Ang II-induced cardiac dysfunction Additionally, VA treatment significantly inhibited the Ang II-induced increase in serum CK-MB levels (Fig. 1J). In conclusion, VA can effectively alleviate Ang II-induced cardiac dysfunction and myocardial injury without affecting blood pressure, thereby mitigating the progression of cardiac dysfunction and cardiac remodeling in mice.

3.2. VA ameliorates Ang II-induced myocardial hypertrophy, inflammatory and myocardial fibrosis

A combination of Western blotting, qRT-PCR, gross cardiac morphological examination, WGA-FITC staining, and analysis of the canonical hypertrophy marker ANP demonstrated that VA treatment significantly attenuated Ang II-induced cardiac hypertrophy in mice. (Fig. 2A–G). Given the established role of chronic inflammation in cardiac remodeling and fibrosis, we investigated whether VA modulates the intra-myocardial inflammatory response. Ang II infusion markedly increased cardiac mRNA abundance of the pro-inflammatory cytokines Il1β, Il6 and Tnf, whereas concomitant VA treatment dose-dependently repressed their expression (Fig. 2H–J). HE staining confirmed these findings. VA markedly alleviated Ang II-induced cardiac structural abnormalities and the increase in cross-sectional area of cardiomyocytes (Fig. 2K). Pathological myocardial hypertrophy is usually accompanied by interstitial fibrosis with diffuse or patchy distribution, specifically manifested as microscar formation and perivascular collagen deposition. Assessment of myocardial fibrosis via Masson's trichrome staining and Sirius red staining showed that VA significantly inhibited collagen fiber deposition and pathological remodeling of the extracellular matrix (Fig. 2L–Q). Collectively, these results demonstrate that VA alleviates Ang II-induced cardiac hypertrophy, inflammation, and fibrosis.

Fig. 2.

VA ameliorates Ang II-induced myocardial hypertrophy, inflammatory and myocardial fibrosis

(A)Representative Western blot analysis results of Myh7, ANP, and BNP in heart tissues. GAPDH was used as the loading control. (B) Quantitative data of the blot intensity of the proteins determined with ImageJ in plane A. (C) mRNA levels of Myh7, Anp and Bnp detected via RT-qPCR in the heart tissues. (D) Representative fresh heart images on a white background [scale bar = 5 mm]. (E) Representative WGA staining image of heart tissues (scale bar = 50 μm). (F) Quantification of WGA immunofluorescence intensity. (G) Immunofluorescence staining of mouse heart tissues for ANP (green), sections were counterstained with DAPI (blue). (H–J) mRNA levels of Il6, Il1b and Tnf detected via RT-qPCR in the heart tissues. (K) Representative H&E staining image of heart tissues (scale bar = 50 μm). (L) Representative Masson staining image of heart tissues (scale bar = 50 μm). (M–N) Quantitative analysis of Masson-Stained Tissue. (O) Representative Sirius Red staining image of heart tissues (scale bar = 50 μm). (P–Q) Quantification of Sirius Red Staining. All quantitative data are presented as mean ± SEM; n = 6; ns = not significant; **p < 0.01 versus the CON group; #p < 0.05, ##p < 0.01 versus the Ang II group, two-tailed Student's t-test.

3.3. VA mitigates heart failure by augmenting mitophagy via the PINK1-Parkin pathway, thereby suppressing NLRP3 inflammasome-mediated pyroptosis

To investigate the potential mechanism of VA against Ang II-induced cardiac injury, we performed RNA-sequencing analysis using the mice heart tissues. 352 significantly different genes were found, including 133 upregulated and 219 downregulated ones (Fig. 3A), when comparing Ang II-treated group to the control group. 313 significantly different genes were identified, including 209 upregulated and 104 downregulated ones, when comparing Ang II-treated group to the VA + Ang II-treated group (Fig. 3B). KEGG enrichment analysis of the differentially expressed genes revealed the mitophagy pathway in both comparisons. Notably, the mitophagy pathway was specifically identified in the Ang II vs drug-treated group (Fig. 3C–F), suggesting that mitochondrial-related pathways may contribute to the cardioprotective effects of VA in this model. Mitophagy has been reported to contribute to the hypertensive HF [18]. Assessment of sample-to-sample correlation demonstrated that all specimens were highly correlated (Fig. 3G). Western blot analysis was examined to determine the pathway of VA-induced mitophagy, identifying the PINK1/Parkin pathway as the underlying mechanism (Fig. 3H–J). To further investigate the molecular mechanisms by which VA ameliorates Ang II-induced HF, key components of the PINK1/Parkin mitophagy pathway—including downstream effectors Beclin1 and LC3-exhibited significantly reduced protein levels, whereas the expression of the mitophagy adapter P62 was markedly increased. Importantly, VA treatment effectively reversed these aberrant protein expression patterns (Fig. 3K–O). Concurrently, we employed immunofluorescence staining to evaluate the effects of VA on mitophagy pathway. Our results demonstrated that Ang II treatment downregulated the expression of PGC-1α (Fig. 3P), indicating that VA alleviates Ang II-induced suppression of mitophagy. Further investigation revealed that Ang II also activated the NLRP3 inflammasome and upregulated the expression of key pyroptosis-related proteins, including NLRP3, ASC, Caspase-1, and GSDMD. Western blot analysis demonstrated that VA treatment significantly suppressed the upregulation of these proteins, indicating that VA also markedly inhibits Ang II-induced pyroptosis (Fig. 3Q–U). In summary, VA attenuates heart failure by enhancing PINK1-Parkin-mediated mitophagy, thereby leading to the subsequent suppression of NLRP3 inflammasome-mediated pyroptosis.

Fig. 3.

VA mitigates heart failure by augmenting mitophagy via the PINK1-Parkin pathway, thereby suppressing NLRP3 inflammasome-mediated pyroptosis

(A-B) Volcano map of differentially expressed genes between Ang II-treated group and control group (A); Volcano map of differentially expressed genes between Ang II-treated group and VA pretreatment + Ang II-treated group (B); Red color indicates upregulation of genes while blue color indicates down regulation, and the grey color in the middle were the genes have no difference. (C–F) KEGG enrichment of the differential gene in Ang II-treated group vs control group (C-D) and Ang II-treated group vs VA pretreatment + Ane II-treated group (E-F). (G) Analysis of sample correlation across the CON, Ang II, and Ang II + Treatment groups. (H) Representative Western blot analysis of Pink1 and Parkin in heart tissues. GAPDH was used as the loading control. (I–J) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in plane H. All quantitative data are presented as mean ± SEM; n = 6; **p < 0.01 versus the CON group; ##p < 0.01 versus the Ang II group, two-tailed Student's t-test.

3.4. VA alleviated Ang II-induced hypertrophy and the inflammatory response, and reduced ROS production and oxidative stress in NRVMs through the promotion of mitophagy

To evaluate whether VA exerts direct therapeutic effects on cardiomyocytes, we utilized NRVMs as an in vitro model. An MTT assay was performed using VA concentrations ranging from 0.1 μM to 80 μM, which allowed us to identify optimal treatment concentrations of 5 and 10 μM (Fig. S2). Cells were pretreated with VA for 1 h followed by incubation with Ang II (1 μM) for either 8 or 24 h. Western blot revealed that VA treatment significantly suppressed the Ang II-induced upregulation of Myh7, ANP, and BNP in a dose-dependent manner (Fig. 4A and B). RT-qPCR data further confirmed that Ang II upregulates the mRNA levels of Myh7, Anp, and Bnp in NRVMs, effects that were markedly attenuated by VA treatment (Fig. 4C). Moreover, rhodamine-phalloidin staining demonstrated that VA significantly alleviated Ang II-induced hypertrophic morphology in NRVMs (Fig. 4D and Fig. S3A). To investigate the pathway through which VA mitigates Ang II-impaired mitophagy, TEM analysis revealed that VA treatment partially restored mitochondrial ultrastructure and mitigated autophagic disruption induced by Ang II (Fig. 4E). We further assessed intracellular ROS generation in NRVMs. Results indicated that Ang II significantly increased ROS levels, contributing to dysfunctional mitophagy, whereas VA treatment markedly reduced green fluorescence intensity, indicating suppression of ROS activity (Fig. 4F). Immunofluorescence staining and Western blot analyses showed that Ang II downregulated the expression of PINK1 and Parkin, while promoting their downstream proteins including P62, LC3, and Beclin1. VA treatment notably reversed these alterations. The lysosomal inhibitor bafilomycin A1 blocks the final step of autophagy—the degradation of autophagic cargo following autophagosome-lysosome fusion. Inhibition of autophagy by this agent leads to increased p62 expression and decreased LC3 expression. However, co-treatment with VA restores the protein levels of both p62 and LC3 to normal. Collectively, these findings indicate that VA promotes mitophagy through activation of the PINK1/Parkin pathway (Fig. 4G–L). Immunofluorescence staining for Parkin further supported this conclusion (Fig. 4M). The mitochondrial membrane potential (ΔΨm), a key indicator of mitochondrial health, was evaluated using JC-1 staining. Ang II treatment resulted in enhanced green fluorescence, reflecting ΔΨm depolarization, which was ameliorated by VA (Fig. 4N and Fig. S3B), demonstrating the ability of VA to preserve mitochondrial function. Therefore, it can be concluded that VA mitigates Ang II-induced hypertrophy, the inflammatory response, and associated oxidative stress and ROS production in NRVMs by promoting mitophagy.

Fig. 4.

VA alleviated Ang II-induced hypertrophy and the inflammatory response, and reduced ROS production and oxidative stress in NRVMs through the promotion of mitophagy

NRVMs were pretreated with 5 or 10 μM VA for l h and then exposed to Ang II (1 μM) for 8 or 24 h. Protein and RNA were extracted to perform immunoblotting and qPCR respectively. (A) Representative Western blot analysis of Myh7, ANP and BNP in the NRVMs. GAPDH was used as the loading control. (B) Quantitative data of the blot intensity of the proteins determined with ImageJ in plane A. (C) RT-qPCR showing mRNA levels of Myh7, Anp and Bnp in the NRVMs. (D) TRITC phalloidin (red) staining of NRVMs showing the effect of VA on Ang II-induced hypertrophic responses. Slides were counterstained with DAPI (blue). (E) Representative TEM images. (F) Representative images of ROS generation. (G) Representative Western blot analysis of PGC-1α, Pink1, Parkin, P62, LC3 and Beclin1 in the NRVMs. GAPDH was used as the loading control. (H–I) Quantitative data of the blot intensity of the proteins determined with ImageJ in plane G. (J) Lysosomal inhibition experiments were performed in NRVMs to assess the expression of P62 and LC3. (K–L) Quantitative data of the blot intensity of the proteins determined with ImageJ in plane J. (M) Immunofluorescence staining of NRVMs for Parkin (red), sections were counterstained with DAPI (blue). (N) Representative images of JC-1 staining and fluorescence intensity (scale bar = 100 μm). All quantitative data are presented as mean ± SEM; n = 3; **p < 0.01 versus the CON group; #p < 0.05, ##p < 0.01 versus the Ang II group, two-tailed Student's t-test.

3.5. By inhibiting the transcriptional regulator BRCA1, which drives the expression of the mitophagy gene PINK1, VA thereby attenuates Ang II-induced pyroptosis in NRVMs

We investigated the ability of VA to bind to the activator site of PINK1. The binding site of VA to PINK1 was further predicted by molecular docking, and simulation analysis showed that Proteins ALA438, LEU413 and ASP362 formed hydrophobic interaction with small molecules, ALA434, TYR431 and ARG361 formed hydrogen bond with small molecules. The molecular docking binding energy between VA and PINK1 was measured at −5.9 kcal/mol (Fig. 5A). VA treatment increases protease susceptibility of the PINK1 in cell lysates as determined by the DARTS assay. DARTS assay demonstrated that VA could directly bind with PINK1 (Fig. 5B and C). CETSA is a broadly applicable biophysical technique allowing detection of changes in ligand/drug binding interactions directly in intact cells. The technique is based on the thermal shift assay (TSA) concept where ligand binding affects protein stability. As shown in Fig. 5D, PINK1 was significantly stabilized in VA-treated cells. As shown in Fig. 5E and Fig. S4A, The thermal stabilization of PINK1 induced by vanillic acid was dose-dependently attenuated by co-incubation with ATP, suggesting that vanillic acid specifically binds to the ATP-binding pocket of PINK1. Taken together, these findings indicate that VA directly binds to PINK1. These findings were corroborated by SPR, which demonstrated that VA could directly bind to PINK1 with a considerable affinity (Fig. 5F). These data suggest that the target protein of VA is PINK1. Furthermore, this investigation also predicted the potentially associated transcription factor (TF) via the RNA-seq analysis, which revealed that 49 genes were modulated by VA (Fig. 5G). The identified 49 genes were subjected to TF perturbations and expression analysis, which indicated a potential involvement of BRCA1, ranked top 1 in the predicted TF list (Fig. 5H). Studies have shown that BRCA1 provides therapeutic benefits in heart failure by safeguarding myocardial cells against apoptosis. To identify the specific binding site of PINK1 within the BRCA1 promoter, various truncated BRCA1 promoter plasmids were constructed. The results indicated that the regulation of BRCA1 levels is determined by the binding of PINK1 to a site located between 4191 and 4272 bp (Fig. 5I and J). Fluorescence co-localization experiments of PINK1 and BRCA1 further demonstrated direct binding between PINK1 and BRCA1 (Fig. 5K). These data suggest that the target protein of VA is PINK1, which subsequently interacts with the transcription factor BRCA1 to mediate its cardioprotective effects. The LDH release data provided robust evidence for the functional assessment of pyroptotic cell death, preliminarily demonstrating that VA exerts a therapeutic effect against Ang II-induced pyroptosis (Fig. 5L). Western blot analysis indicated that Ang II stimulation markedly upregulated NLRP3 protein expression, suggesting activation of the NLRP3 inflammasome. Consistent with this activation, the expression levels of downstream proteins, including ASC, Caspase-1, and GSDMD, were also elevated. BRCA1 knockout attenuated this increase, and subsequent VA treatment led to a further reduction in protein levels, indicating that they synergistically suppress NLRP3 inflammasome-induced pyroptosis (Fig. 5M–Q). Consistently, RT-qPCR results showed that Ang II increased the mRNA levels of key inflammatory cytokines released upon NLRP3 inflammasome activation. These levels were reduced in both the BRCA1 knockout group and the group receiving combined knockout and VA treatment (Fig. 5R–T). NLRP3 immunofluorescence staining further confirmed that both BRCA1 knockout and its combination with VA regulated the Ang II-induced fluorescence intensity of NLRP3 in cardiomyocytes (Fig. 5U). Flow cytometric assessment of apoptosis revealed a gradual reduction in the apoptotic rate in both the knockout group and the knockout-plus-treatment group (Fig. 5V). Collectively, these results establish a critical role for BRCA1 in heart failure pathogenesis driven by NLRP3 inflammasome-derived inflammatory factors, and demonstrate that VA ameliorates this condition by attenuating the BRCA1-mediated pathway.

Fig. 5.

By inhibiting the transcriptional regulator BRCA1, which drives the expression of the mitophagy gene PINK1, VA thereby attenuates Ang II-induced pyroptosis in NRVMs

(A) Molecular docking of VA with BRCA1. (B) VA treatment increases protease susceptibility of the BRCA1 in cell lysates as determined by the DARTS assay. (C) Quantitative data of the blot intensity of corresponding proteins determined by Image J software. (D) NRVMs cells were treated with 6 μM VA for 2 h and subsequently heated at different temperatures for 3 min. After freeze-thaw cycles for cell lysis, the soluble BRCA1 protein levels were examined by Western blotting. (E) Competition experiments between VA and ATP for PINK1. (F) SPR analysis between VA and BRCA1. (G) Venn diagram of up-regulated genes of in Ang II-treated group vs control group and Ang II-treat group vs VA pretreatment + Ang II-treated group. (H) Top list of TF Perturbations followed by expression analysis of intersecting genes in plane G. (I–J) Relative luciferase activity was detected in NRVMs cells cotransfected with a luciferase reporter plasmid containing the various truncated BRCA1 promoter sequence and a PINK1 overexpression plasmid. (K) Fluorescence co-localization staining of PINK1 and BRCA1. (L) LDH release assay. (M) Following BRCA1 knockdown in NRVMs cells, cells were pre-treated with or without 10 μM VA for 1 h before exposure to Ang II (1 μM) for 2 h. Representative Western blot analysis of NLRP3, ASC, Caspase1 and GSDMD are shown with GAPDH used as the loading control. (N–Q) Quantitative data of the blot intensity of corresponding proteins determined by Image J software in plane J. (R–T) mRNA levels of Il6, Il1b and Tnf detected via RT-qPCR in the NRVMs. (U) Immunofluorescence staining of NRVMs for NLRP3 (red), sections were counterstained with DAPI (blue). (V) The rate of apoptosis in NRVMs was quantified by flow cytometric analysis following Annexin V/PI iodide staining. All quantitative data is presented as Mean ± SEM; n = 3; **p < 0.01; ns = not significant, two-tailed Student's t-test.

4. Discussion

Therapeutic benefits of VA have been observed in models including arthritis, liver fibrosis, and muscle atrophy, with evidence suggesting that the modulation of mitochondrial function underlies its efficacy. Our in vivo and in vitro studies demonstrate that VA suppresses NLRP3 inflammasome-mediated heart failure by restoring cardiomyocyte mitochondrial function through the PINK1- BRCA1 signaling axis (Graphical Abstract). These findings provide novel mechanistic insights and a potential therapeutic strategy for HF management.

Mitophagy is an autophagy-mediated lysosomal degradation process that specifically removes damaged or unnecessary mitochondria, and it represents the most well-studied type of selective autophagy [19]. In recent years, studies have indicated that mitochondrial dysfunction plays a critical role in the pathogenesis of HF [20]. As the primary energy source for cardiomyocytes, mitochondria are central organelles for maintaining cardiac function; the efficient degradation of dysfunctional mitochondria is essential for preserving cellular function, and insufficient mitophagy ultimately leads to mitochondrial dysfunction and the progression of HF [21]. Currently, Parkin-dependent ubiquitin-mediated mitophagy is the most extensively characterized mitophagic pathway, consisting of three key components: a mitochondrial damage sensor (PINK1), a signal amplifier (Parkin), and a signal effector (ubiquitin chain) [22]. PINK1 is a serine/threonine kinase localized to the mitochondrial outer membrane. Its integration into mitochondria is facilitated by the coordinated action of translocases of the inner and outer membranes. Upon mitochondrial damage, PINK1 undergoes autophosphorylation and recruits Parkin, which subsequently interacts with p62 and LC3 [23]. Beclin1, LC3, and p62 are key mediators of mitophagy and are widely used as markers to monitor and quantify autophagic activity under both physiological and pathological conditions. Beclin1 is essential for the initiation of mitophagy by interacting with various cofactors to trigger the autophagic protein cascade. LC3 serves as a reliable indicator of autophagic flux, mediating membrane elongation and autophagosome maturation through its binding to P62 [24]. Additionally, P62 facilitates the transport of ubiquitinated proteins for degradation via mitophagy [25]. Our experimental results demonstrate that Ang II induction led to significant alterations in key mitophagy markers in cardiac tissue: expression levels of PINK1, Parkin, LC3, and Beclin1 were downregulated, while p62 was upregulated. These findings are consistent with previously reported data in studies of myocardial injury [26]. PINK1 promotes the translocation of Parkin to the mitochondrial outer membrane, facilitating the formation of mitophagosomes [27]. Previous studies have shown that VA can alleviate H₂O₂-induced damage in H9c2 cells by regulating the PINK1/Parkin pathway [10]. Our experimental results confirm that VA treatment upregulates the expression of PINK1 and Parkin, indicating its role in promoting mitophagy. Experiments such as molecular docking, DARTS, CETSA, and SPR verified the interaction between VA and PINK1.

Pyroptosis is a form of programmed cell death mediated by canonical and non-canonical pathways, which ultimately leads to the lysis and rupture of the cell membrane as well as the release of pro-inflammatory cytokines, thereby triggering a robust inflammatory response [28]. The NLRP3 inflammasome, a member of the NOD-like receptor (NLR) family, is activated through the assembly of NOD-like receptor pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), and pro-caspase-1. By activating Caspase-1 and releasing interleukin-1β (IL-1β) and interleukin-18 (IL-18), the NLRP3 inflammasome promotes cardiac hypertrophy [29]. Dysfunctional mitophagy and the accumulation of oxidative stress products are critical pathways leading to pyroptosis [30]. The dissipation of mitochondrial membrane potential and the release of mitochondrial damage-associated molecular patterns (mtDAMPs, e.g., mitochondrial ROS) result in the activation of the NLRP3 inflammasome. Therefore, mitochondrial dysfunction plays a central role in NLRP3 inflammasome activation through multiple mechanisms [31]. Studies have shown that ROS generation contributes to NLRP3 inflammasome-mediated pyroptosis and plays a role in the pathogenesis of various inflammatory diseases, including HF. Ang II induction leads to increased ROS production [32], resulting in mitochondrial dysfunction, which is a key contributor to NLRP3 inflammasome activation [33]. Furthermore, Ang II upregulates the expression of key components of the NLRP3 inflammasome pathway—including NLRP3, ASC, Caspase-1, and IL-1β—thereby activating caspase-1 and triggering pyroptosis. Subsequent analysis of transcription factors uncovered that BRCA1 is engaged in the process through which VA alleviates pathological cardiac hypertrophy via the mitophagy pathway. Evidence suggests that BRCA1 is a potential biomarker for HF [34]. We found that the regulation of BRCA1 levels is determined by the binding of PINK1 to a site located between 4191 and 4272 bp. Next, to investigate its functional role, we silenced BRCA1 and assessed its impact on NLRP3 inflammasome-mediated pyroptosis. Our experimental data, encompassing Western blotting, flow cytometry, and PCR analyses, consistently demonstrated that BRCA1 knockout downregulated the expression of pyroptosis-related markers (NLRP3, ASC, Caspase-1, and GSDMD). The most pronounced reduction in protein expression was achieved with the combined intervention of BRCA1 knockout and VA treatment, collectively supporting our hypothesis that VA suppresses NLRP3 inflammasome-mediated heart failure by restoring cardiomyocyte mitochondrial function through the PINK1- BRCA1 signaling axis.

Through modern pharmacological approaches, this study elucidates the multi-target mechanisms of a natural compound derived from traditional medicine. Our study, which integrates cell biology and pharmacological techniques, revealed that VA counteracts Ang II-induced heart failure by inhibiting cardiomyocyte pyroptosis through the activation of the PINK1-mediated mitophagy pathway and BRCA1. These results provide a possible treatment approach for heart failure and reveal a new heart-protective mechanism through the coordinated control of mitophagy and ferroptosis.

Several limitations of this study should be acknowledged. All in vitro experiments were performed only in neonatal rat ventricular myocytes without validation in adult or human-derived cardiomyocytes, which limits translational relevance. Only male mice were used, so the findings may not extend to female patients. The treatment duration was relatively short, and long-term outcomes such as survival were not assessed. A comprehensive evaluation of off-target effects is lacking. Additionally, only the Ang II infusion model (hypertensive HFpEF) was used, so whether the PINK1-BRCA1-NLRP3 pathway exerts similar protective effects in ischemic or other forms of heart failure remains unknown. These limitations have been discussed with reasoned interpretations of their potential impact on the experimental results.

Based on our experimental findings and the limitations discussed above, the following implications and future directions emerge. Specifically, the PINK1-BRCA1 regulatory axis identified in this study may have broader pathophysiological relevance beyond heart failure and therefore warrants exploration in other disease models involving mitochondrial dysfunction and inflammation. Additionally, while our experiments were performed in H9c2 cells and primary neonatal rat cardiomyocytes, key phenotypes should be validated in adult rat ventricular myocytes (ARVMs), which more closely recapitulate the contractile and electrophysiological properties of the mature heart, as well as in human-derived cardiomyocytes, to assess the regulatory role of vanillic acid on the PINK1-BRCA1-NLRP3 axis in the human context. Furthermore, all pivotal animal experiments, including heart failure models following transverse aortic constriction or myocardial infarction, should be repeated in female mice to evaluate potential sex differences in cardiac function, survival, and inflammatory pathway responses, as our current study was conducted only in male mice. Finally, to further characterize the molecular basis of vanillic acid's action, kinase profiling should be performed to evaluate the binding affinity or inhibitory activity of vanillic acid against a panel of over 300 human kinases, which could reveal additional targets and off-effects relevant to its therapeutic potential.

Author contributions

Mengyang Wang: Writing – original draft, Writing – review and editing, Data curation, Funding acquisition, Project administration, Resources. Chang Liu: Software, Methodology, Formal analysis. Linxin Zhang: Software, Methodology. Zhuoqun Wang: Validation, Visualization. Shuang Yan: Writing – review and editing, Investigation, Resources, Supervision.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.