What this is
- Hopeaphenol, a tetramer of resveratrol, shows promise in combating by activating .
- This study investigates hopeaphenol's protective effects through both in vivo and in vitro experiments.
- Findings suggest that hopeaphenol improves cardiac function and mitochondrial metabolism while reducing oxidative stress.
Essence
- Hopeaphenol alleviates induced by pressure overload through activation, enhancing mitochondrial function and reducing oxidative stress.
Key takeaways
- Hopeaphenol improves left ventricular ejection fraction and fractional shortening in TAC mice, indicating enhanced cardiac function.
- Hopeaphenol significantly reduces and fibrosis in TAC mice, restoring cardiomyocyte morphology and reducing collagen deposition.
- Hopeaphenol's cardioprotective effects are mediated by activation, confirmed through direct binding and functional assays.
Caveats
- The study relies on the TAC model, which may not fully represent all forms of .
- Future studies are needed to validate the role of and explore other potential pathways involved.
- The use of Compound C as an inhibitor may have off-target effects that complicate interpretations.
Definitions
- Cardiac hypertrophy: An increase in heart muscle mass due to pressure or volume overload, which can lead to heart failure.
- AMPK: AMP-activated protein kinase, a key regulator of cellular energy homeostasis and metabolism.
Simplified
1. Introduction
Cardiac hypertrophy represents an adaptive response of the heart to conditions of pressure or volume overload. Initially, this adaptation can preserve cardiac function; however, sustained overload results in pathological remodeling, which is characterized by myocardial fibrosis, arrhythmias, and ultimately progression to heart failure [1,2,3,4]. Disruptions in energy metabolism play a crucial role in the onset and progression of cardiac hypertrophy. A fundamental driving factor is the heart's high energy demand, which relies on ATP production through mitochondrial oxidative phosphorylation [5]. Disorders in cardiac energy metabolism often precede the development of hypertrophy [6]. Increased cardiac load disrupts mitochondrial homeostasis, leading to excessive production of reactive oxygen species (ROS) [7]. The accumulation of ROS exacerbates disturbances in energy metabolism by activating pro-inflammatory and pro-apoptotic signaling pathways, thereby accelerating the transition from cardiac hypertrophy to heart failure [8,9,10].
AMP-activated protein kinase (AMPK), a critical regulator of energy metabolism, is activated in response to pressure overload and plays an essential role in maintaining mitochondrial homeostasis by modulating downstream target proteins, thereby exerting a significant influence on cardiac metabolism [11,12,13]. Although the natural polyphenol resveratrol is known to ameliorate oxidative stress, its clinical application is constrained by its low bioavailability and adverse effects at high doses, such as gastrointestinal discomfort and thyroid dysfunction [14,15,16,17]. Previous studies have shown that hopeaphenol, a resveratrol tetramer, exhibits cardioprotection-related activities—including anti-apoptosis and mitochondrial function regulation—and displays functional differences from other resveratrol tetramers [18]; however, its effects on cardiac hypertrophy and the underlying mechanism of AMPK modulation remain unclear.
Considering the potential of hopeaphenol in modulating mitochondrial function and the pivotal role of AMPK in energy metabolism, this study posits that hopeaphenol may alleviate pressure overload-induced cardiac hypertrophy by activating the AMPK signaling pathway, with mechanisms associated with the amelioration of mitochondrial metabolic abnormalities. This hypothesis was validated through in vivo and in vitro experiments to identify a novel therapeutic target for the prevention and treatment of heart failure. Furthermore, this investigation elucidates the structure-activity relationship of natural polyphenols, highlights the advantages of tetrameric structures over monomeric forms, and provides a theoretical framework for addressing the limitations of existing pharmacological agents, such as the dosage constraints associated with resveratrol. Consequently, this research holds significant implications for both fundamental studies and clinical applications.
2. Materials and Methods
2.1. Experimental Materials
2.1.1. Experimental Animals
Male C57BL/6 mice (wild-type, specific pathogen-free [SPF]) aged 6–8 weeks (weight: 20–25 g) were used in this study. The mice were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine. All animals were housed in the SPF-grade animal facility of Guangzhou University of Chinese Medicine under controlled conditions (temperature: 22 ± 2 °C, humidity: 50% ± 10%, 12 h light-dark cycle) with ad libitum access to standard laboratory chow and water. Environmental enrichment (nesting materials and plastic shelters) was provided in each cage for the mice. The animal study was reviewed and approved by the Ethics Committee of Guangzhou University of Chinese Medicine (Approval No. XS20240031, approved on 19 March 2024).
2.1.2. Cell Line
The HL-1 mouse cardiomyocyte cell line was purchased from iCellverse and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin in a 37 °C, 5% CO2 incubator.
2.1.3. Main Reagents, Consumables, and Instruments
Hopeaphenol (≥98%, #388582-37-4), was purchased from Sigma-Aldrich (St. Louis, MO, USA). Compound C (#S7840) and Angiotensin II (Ang II, #HY-P0108) were obtained from Selleck Chemicals (Houston, TX, USA) and MedChemExpress (MCE, Monmouth Jct., NJ, USA), respectively. The related antibodies used in this study included anti-phospho-AMPK (pAMPK, #83924-1-RR, Proteintech Group, Rosemont, IL, USA), anti-AMPK (#AB32047, Abcam, Cambridge, UK), and anti-SIRT1 (#DF6033, Affinity Biosciences, Cincinnati, OH, USA). Detection kits were sourced as follows: Superoxide Dismutase (SOD) assay kit (#BC5165, Solarbio Life Sciences, Beijing, China); Adenosine Triphosphate (ATP) assay kit (#S0026, Beyotime Biotechnology, Shanghai, China); Reactive Oxygen Species (ROS) assay kit (#S0033S, Beyotime Biotechnology, Shanghai, China); and JC-1 mitochondrial membrane potential assay kit (#C2006, Beyotime Biotechnology, Shanghai, China).Instruments included the ChemiScope S6 imager, ViiA 7 real-time PCR system, Varioskan LUX microplate reader, etc.
2.2. Experimental Methods
2.2.1. Animal Model Construction and Grouping
Thirty-six C57BL/6 mice were randomly assigned to six experimental groups (n = 6 per group) using a computer-generated random number sequence. The specific grouping and treatment protocols for each group were as follows: (1) Sham group: Mice underwent thoracotomy without aortic ligation; (2) Transverse Aortic Constriction (TAC) model group: Cardiac hypertrophy was induced via TAC surgery [19]; (3) Hopeaphenol (HP) low-dose group: Mice received an intraperitoneal injection of 5 mg/kg HP after TAC surgery; (4) HP medium-dose group: Mice received an intraperitoneal injection of 10 mg/kg HP after TAC surgery; (5) HP high-dose group: Mice received an intraperitoneal injection of 20 mg/kg HP after TAC surgery; and (6) Metformin group: Mice received an intraperitoneal injection of 200 mg/kg metformin after TAC surgery.
The dosage of HP was determined based on preliminary experiments. These pre-experiments showed that doses below 5 mg/kg exerted minimal cardioprotective effects, while doses exceeding 20 mg/kg failed to enhance therapeutic efficacy and instead led to increased drug exposure. In accordance with standard TAC model protocols, 200 mg/kg metformin was used as a positive control. This dose was selected based on: (1) species-specific pharmacokinetics—rodents (especially mice) have higher metabolic rates and faster drug clearance than humans, requiring higher mg/kg doses to achieve comparable pharmacologically active exposure; (2) established efficacy in TAC models, as 200 mg/kg (intraperitoneal injection) is widely validated for demonstrating metformin's AMPK-mediated cardioprotection [20,21]. Drug administration was initiated 4 weeks after TAC surgery and continued for an additional 4 weeks. This timing was intentionally chosen to investigate hopeaphenol's potential for therapeutic reversal of established cardiac hypertrophy and dysfunction (rather than prevention), as: (1) clinically, patients typically seek treatment after symptom onset and disease establishment, making a reversal strategy more translationally relevant; (2) allowing hypertrophy to fully develop before intervention ensures observed improvements are attributed to direct therapeutic effects of hopeaphenol, not prevention of initial adaptive responses. The Sham group and TAC model group were not given any drugs; instead, they received intraperitoneal injections of an equal volume of normal saline.
2.2.2. Inclusion and Exclusion Criteria
All male C57BL/6 mice (6–8 weeks old, 20–25 g) undergoing transverse aortic constriction (TAC) surgery were included in the study. No animals were excluded from the analysis prior to the experimental endpoint (8 weeks post-TAC, including 4 weeks of drug administration) unless specified otherwise. Mice that died during TAC surgery or within the first 48 h post-operation (n = 2) were classified as surgical fatalities (not related to experimental intervention) and were replaced with age- and weight-matched mice to maintain the predetermined group size (n = 6 per group). Data points from all surviving mice were included in the final statistical analysis, and no data were excluded post hoc, in accordance with the predefined experimental protocol.
2.2.3. Hopeaphenol Solution Preparation
Hopeaphenol was dissolved in DMSO, mixed according to a ratio of DMSO:PEG-300:normal saline = 1:8:11, stored at 4 °C in the dark, and confirmed to be free of precipitation before use.
2.2.4. Cardiac Function Detection
At the end of the experiment, detection was performed using a small animal ultrasound imaging system. Mice were anesthetized with isoflurane (2%, oxygen flow rate: 1.5 L/min), and hemodynamic indices such as left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured.
2.2.5. Tissue Staining
The left ventricular tissues were fixed, and hematoxylin and eosin (HE) staining was used to observe cardiomyocyte morphology. Masson staining was used to evaluate fibrosis, and WGA staining combined with ImageJ (Fiji distribution, version 2.15.0) was used to measure cardiomyocyte cross-sectional area (five random fields per section).
2.2.6. Western Blot
Total proteins from tissues or cells were extracted and quantified using the BCA method. The proteins were then subjected to SDS-PAGE, transferred to PVDF membranes, and blocked. Following this, the membranes were incubated with primary antibodies AMPK, pAMPK, and SIRT1) and secondary antibodies. The proteins were detected using chemiluminescence, and protein expression was analyzed.
2.2.7. qRT-PCR Detection
RNA was extracted and reverse-transcribed into cDNA. The SYBR Green method was used for amplification, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal reference. The relative expression levels of target genes (AMPK, SIRT1, and others) were calculated using the 2−ΔΔCt method (primer sequences are shown in Table 1).
2.2.8. CCK-8 Assay for Cell Viability
HL-1 cells were seeded in 96-well plates (5 × 104 cells/mL), cultured for 24 h, and then treated with hopeaphenol at concentrations of 1 μM, 5 μM, 10 μM, 20 μM, and 50 μM, as well as Compound C, for 12 h, with a blank control group included. Subsequent procedures were performed according to the instructions.
2.2.9. Cell Modeling, Grouping, and Administration
HL-1 cells were pretreated with hopeaphenol (1–50 μM) for 2 h, and then induced to undergo hypertrophy by 1 μM Ang II for 24 h. In inhibition groups, 10 μM Compound C was added 1 h prior to hopeaphenol treatment.
Dose rationale: HP and CC concentrations (1–50 μM) were determined via CCK-8, with 1–10 μM as a non-toxic range (no significant viability reduction), referencing stilbene use in cardiomyocyte studies (1–20 μM for anti-hypertrophy). Ang II (1 μM) [22] and Compound C (10 μM) followed standard protocols for inducing hypertrophy and inhibiting AMPK.
2.2.10. Mitochondrial ROS Detection
HL-1 cells were seeded in culture plates and cultured to an appropriate density, then incubated with 20 μM DCFH-DA fluorescent probe in the dark for 30 min. After washing with PBS to remove excess probe, detection was performed by fluorescence microscopy.
2.2.11. Mitochondrial Membrane Potential Detection
Mitochondrial membrane potential was detected by JC-1 staining. Treated cardiomyocytes were incubated with JC-1 working solution for 20 min, washed three times with PBS, and the intensities of red and green fluorescence were observed under a fluorescence microscope. The ratio of red to green fluorescence intensity was calculated to evaluate changes in mitochondrial membrane potential.
2.2.12. Cell Thermal Shift Assay (CETSA)
Cells were incubated with 10 μM hopeaphenol for 2 h, then heated at a gradient of 37–65 °C. The residual amount of AMPK protein was detected by Western blotting.
2.3. Network Pharmacology Analysis
2.3.1. Screening of Active Components and Targets
Hopeaphenol was obtained from the TCMSP database and screened according to the criteria of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. The results were converted to human gene names via the UniProt database.
2.3.2. Acquisition of Heart Failure Target Genes
The term "Heart Failure" was employed to search the OMIM and GeneCards databases. The data retrieved from these sources was consolidated, and any duplicate entries were eliminated.
2.3.3. Screening of Intersection Targets
A comparative analysis between the targets of hopeaphenol and those associated with heart failure was conducted using the EVenn (version 3.0.0) tool, resulting in the identification of potential therapeutic targets at the intersection.
2.3.4. Construction of Hopeaphenol-Heart Failure Target Network
The intersection targets identified between hopeaphenol and heart failure were imported into Cytoscape version 3.9.1. In this network, node degree values were utilized to indicate the strength of the associations.
2.3.5. Construction of PPI Network and Screening of Core Targets
Intersection targets were imported into the STRING database (species: Homo sapiens, confidence threshold > 0.9, with disconnected nodes excluded). Subsequently, the data were imported into Cytoscape software, where topological parameters were calculated to identify core targets.
2.3.6. GO and KEGG Enrichment Analyses
Potential targets underwent enrichment analysis using the DAVID database. The results were filtered based on a predetermined p-value threshold and visualized accordingly.
2.3.7. Molecular Docking Analysis
The structure of AMP-activated protein kinase (AMPK) was retrieved from the Protein Data Bank (PDB). For docking analysis, we selected two structures: the crystal structure of the human AMPK α2 kinase domain (PDB ID: 2H6D, the predominant cardiac catalytic isoform) and the full-length active-state AMPK heterotrimer (PDB: 6B2E). After optimizing the hopeaphenol structure, AutoDock Vina version 1.2.5 was used to calculate binding energy and assess complex stability. We note that 2H6D only includes the kinase domain (no β/γ regulatory subunits), so these results preliminarily explore catalytic site interactions; future studies will investigate allosteric modulation via β/γ subunits.
2.4. Statistical Analysis
Experimental data were analyzed using GraphPad Prism 9.5 software. Comparisons among multiple groups were performed by one-way analysis of variance (one-way ANOVA), and pairwise comparisons between groups were performed using the LSD or Dunnett's method. A p-value is significant.
3. Results
3.1. Hopeaphenol Significantly Enhances Cardiac Function in TAC Mice
Compared to the Sham group, the TAC group exhibited a marked reduction in left ventricular ejection fraction (LVEF, Figure 1C) and left ventricular fractional shortening (LVFS, Figure 1D), alongside a significant increase in left ventricular end-diastolic dimension (LVIDd, Figure 1E), left ventricular end-systolic dimension (LVIDs, Figure 1F), left ventricular posterior wall thickness at end-diastole (LVPWd, Figure 1G), and left ventricular posterior wall thickness at end-systole (LVPWs, Figure 1H). These findings confirm the successful modeling of TAC and the induction of cardiac hypertrophy and dysfunction. In contrast, the cardiac function-related indices in the hopeaphenol treatment groups demonstrated significant improvements in a dose-dependent manner, with the high-dose group exhibiting the most pronounced effects, comparable to those observed in the metformin group (Figure 1).
3.2. Hopeaphenol Mitigates Cardiac Hypertrophy and Fibrosis in TAC Mice
In comparison to the Sham group, the TAC group exhibited a significant increase in heart volume (Figure 2B), left ventricular mass (LV Mass, Figure 2C), heart weight-to-body weight ratio (Figure 2D), and heart weight-to-body surface area ratio (Figure 2E). Hematoxylin and eosin (HE) staining revealed that cardiomyocytes in the TAC group were markedly hypertrophic and exhibited morphological disarray. In contrast, the degree of cardiomyocyte hypertrophy in the hopeaphenol treatment groups was significantly diminished, with morphology appearing more regular (Figure 2F). Masson staining indicated a significant increase in collagen fiber deposition and exacerbated fibrosis within the myocardial tissue of the TAC group, whereas cardiac fibrosis was mitigated in the hopeaphenol treatment groups (Figure 2G,H). Wheat germ agglutinin (WGA) staining demonstrated a significant enlargement in the cross-sectional area of cardiomyocytes in the TAC group, with hypertrophy being alleviated in the hopeaphenol treatment groups (Figure 2I,J). Furthermore, Western blot and quantitative PCR analyses revealed that the protein and mRNA expression levels of BNP and β-MHC in the myocardial tissue of the TAC group were significantly elevated, whereas these levels were reduced in the hopeaphenol treatment group (Figure 2K–O). The aforementioned findings suggest that hopeaphenol ameliorates TAC-induced cardiac hypertrophy and fibrosis in a dose-dependent fashion, exhibiting effects comparable to those observed in the metformin treatment group (Figure 2).
3.3. Hopeaphenol Alleviates Oxidative Stress and Metabolic Issues
In the TAC group, myocardial tissue showed significant oxidative stress and metabolic issues. Superoxide dismutase (SOD) activity (Figure 3A), crucial for combating reactive oxygen species (ROS), was notably reduced, indicating diminished oxidative stress defenses. Concurrently, serum adenosine triphosphate (ATP) levels (a marker of systemic energy metabolism, Figure 3B), vital for reflecting whole-body metabolic status, dropped significantly, while serum adenosine diphosphate (ADP) levels (Figure 3C) rose, pointing to a systemic metabolic disturbance in TAC mice. Additionally, malondialdehyde (MDA), a systemic oxidative stress indicator, was significantly elevated (Figure 3D). Hopeaphenol treatment reduced these issues in a dose-dependent way by boosting SOD activity, enhancing antioxidant defenses, lowering ROS levels, increasing serum ATP production, and decreasing ADP accumulation, thereby restoring energy balance. To directly assess cardiac mitochondrial function, in vitro experiments (Section 3.6) measured mitochondrial membrane potential and ROS in HL-1 cells, providing direct evidence of HP's beneficial effects on cardiac mitochondrial function (Figure 3).
3.4. Hopeaphenol Exerts Cardioprotective Effects by Targeting the AMPK Pathway
Network pharmacology analysis revealed that there are 67 common targets shared between hopeaphenol and heart failure, representing 62.61% of the total targets of hopeaphenol (Figure 4A). Protein–protein interaction network analysis demonstrated that these targets constitute a highly interconnected module, with an average connectivity of 13.156 and a density of 0.209. Within this network, key downstream effectors of AMPK, such as CCND1 (connectivity = 28), MTOR (connectivity = 26), and AKT2 (connectivity = 13), were identified as central components (Figure 4B). KEGG pathway enrichment analysis further corroborated the significant activation of the AMPK signaling pathway (p < 0.05), with 60% of the annotated molecules in this pathway represented within the network (Figure 4C). Molecular docking was performed using two AMPK structures: the human AMPK α2 kinase domain (PDB ID: 2H6D, predominant cardiac catalytic isoform) and the full-length active-state AMPK heterotrimer (PDB: 6B2E). Results showed hopeaphenol had strong binding affinity to the active site of AMPK α2 (PDB ID: 2H6D) with a binding energy of −9.206 kcal/mol, forming stable hydrogen bonds with key residues (GLU-143 and ASP-139) (Figure 4D,E). Notably, hopeaphenol exhibited weaker binding affinity to the full-length active-state AMPK (PDB: 6B2E, binding energy: −8.083 kcal/mol), indicating a preference for the inactive kinase state.
To verify the direct interaction between Hopeaphenol (HP) and AMPK, we performed a Cellular Thermal Shift Assay (CETSA) combined with Western blot analysis. First, we assessed the thermal stability of phospho-AMPK (pAMPK): pAMPK protein is susceptible to degradation within the temperature range of 46–69 °C; in the DMSO control group, pAMPK degradation initiated at 59.5 °C, while in the HP-treated group, pAMPK degradation was significantly attenuated at this temperature (Figure 4F). To rule out the possibility that the signal shift reflected only phospho-epitope stabilization, we further detected the thermal stability of total AMPK using an antibody independent of phosphorylation status. The results showed that Hopeaphenol treatment induced a highly significant increase in the thermal stability of total AMPK protein, with a ΔTm of +4.79 °C compared to the DMSO control (p < 0.05), while the ΔTm for pAMPK was +0.91 °C (Figure 4G,H). This differential stabilization pattern was consistent with molecular docking results, collectively confirming HP's state-dependent binding preference for inactive AMPK. Western blot analysis revealed that in the TAC model, myocardial pAMPK levels were significantly reduced while total AMPK expression remained unchanged; the AMPK downstream effector SIRT1 was also downregulated (Figure 4J–L). Notably, hopeaphenol treatment reversed these alterations in a dose-dependent manner, with pAMPK and SIRT1 levels increasing with rising HP concentrations (Figure 4J–L).
These findings demonstrate that hopeaphenol directly interacts with AMPK (preferentially the inactive state), thereby enhancing its stability, promoting its phosphorylation, and potentially activating the AMPK/SIRT1 signaling pathway (Figure 4).
3.5. Hopeaphenol Mitigates Ang II-Induced Cardiomyocyte Hypertrophy Via Activation of the AMPK/SIRT1 Pathway
The CCK-8 assay was employed to ascertain the safe concentration range of hopeaphenol (1–10 μM), within which cell viability did not significantly differ from that of the normal control group (Figure 5A,B). Based on these findings, three concentrations—1 μM (low), 5 μM (medium), and 10 μM (high)—were selected for subsequent mechanistic investigations. Western blot analysis revealed that Ang II treatment markedly suppressed the expression of pAMPK and SIRT1 proteins in cardiomyocytes, which was accompanied by an upregulation of the cardiac hypertrophy marker BNP (Figure 5D–G). Treatment with hopeaphenol reversed these alterations in a dose-dependent manner, with expression levels of pAMPK and SIRT1 in the 10 μM hopeaphenol treatment group being 3.68-fold and 1.84-fold higher, respectively, compared to the Ang II group. Cellular immunofluorescence demonstrated that hopeaphenol treatment alone significantly enhanced the fluorescence signals of pAMPK and SIRT1. However, the concurrent application of the AMPK inhibitor Compound C completely abrogated these effects of hopeaphenol, resulting in pAMPK and SIRT1 expression levels reverting to those observed in the Ang II group (Figure 5H–K). Furthermore, phalloidin staining demonstrated that hopeaphenol treatment markedly decreased the surface area of cardiomyocytes, and this anti-hypertrophic effect was entirely negated by the presence of Compound C (Figure 5L–M). These findings indicate that the cardioprotective effect of hopeaphenol is contingent upon the activation of the AMPK/SIRT1 signaling pathway (Figure 5).
3.6. Hopeaphenol Mitigates Ang II-Induced Mitochondrial Dysfunction in HL-1 Cardiomyocytes Via the AMPK Pathway
Stimulation with Ang II markedly elevated mitochondrial reactive oxygen species (ROS) levels in HL-1 cardiomyocytes (Figure 6A,B) and led to a reduction in mitochondrial membrane potential (Figure 6C,D). Treatment with hopeaphenol significantly counteracted these aberrant alterations by decreasing mitochondrial ROS levels and restoring membrane potential. Notably, pre-treatment with the AMPK-specific inhibitor, Compound C, entirely abrogated the beneficial effects of hopeaphenol on both mitochondrial ROS levels and membrane potential. These findings substantiate that hopeaphenol ameliorates Ang II-induced mitochondrial oxidative stress and functional impairment through the activation of the AMPK pathway (Figure 6).
3.7. Inhibition of AMPK Activity Partially Blocks the Protective Effects of Hopeaphenol on Cardiomyocytes
Western blot analyses revealed that Angiotensin II (Ang II) treatment markedly suppressed the protein expression of AMPK downstream effectors, PGC-1α and SIRT1, in HL-1 cardiomyocytes. Concurrently, Ang II treatment led to an upregulation of cardiac hypertrophy markers, BNP and β-MHC. Intervention with hopeaphenol significantly ameliorated these aberrant changes. However, pre-treatment with Compound C entirely negated the effects of hopeaphenol (Figure 7A–E). Further, qPCR analysis corroborated that Ang II significantly reduced SIRT1 mRNA levels while elevating the mRNA expression of BNP and β-MHC. Hopeaphenol treatment substantially improved these parameters, but pre-treatment with Compound C completely nullified hopeaphenol's protective effects (Figure 7F–H). Additionally, hopeaphenol intervention significantly mitigated the Ang II-induced increase in the BAX/BCL2 ratio and the elevation of Cleaved caspase-3 protein levels. This anti-apoptotic effect of hopeaphenol was entirely obstructed by pre-treatment with Compound C (Figure 7I–K). The aforementioned findings suggest that hopeaphenol exerts protective effects against cardiac hypertrophy and apoptosis primarily by activating the AMPK/SIRT1 signaling pathway, though the involvement of other potential pathways cannot be completely excluded due to the off-target effects of Compound C. Notably, the inhibition of AMPK activity substantially abrogates the cardioprotective effects of hopeaphenol, which strongly supports the critical role of AMPK in mediating these beneficial effects (Figure 7).
4. Discussion
This study demonstrates that hopeaphenol alleviates pressure overload-induced cardiac hypertrophy and delays heart failure progression by activating the AMPK signaling pathway (with direct binding supported by CETSA and docking), which enhances mitochondrial energy metabolism and reduces oxidative stress. Below, we elaborate on the pathological mechanisms underlying cardiac hypertrophy and the specific role of hopeaphenol in modulating these processes.
Cardiac hypertrophy, a pathological remodeling response to overload, is characterized by a vicious cycle involving abnormal mitochondrial energy metabolism and oxidative stress [3,23,24]. This pathological link is further supported by clinical research in human hypertrophic cardiomyopathy: studies have shown mitochondrial dysfunction is closely associated with structural disruptions in cardiomyocytes, and this dysfunction can be partially reversed by enhancing NADH-driven mitochondrial respiration—highlighting the therapeutic potential of targeting mitochondrial metabolism in hypertrophy [25]. It is initiated by cardiomyocytes sensing and responding to mechanical load, in which mechanical stimuli are converted into biochemical signals Via mechanotransduction. In the early stages, adaptive changes in myocardial structure and function can maintain cardiac pumping efficiency [24]; however, sustained or excessive load triggers maladaptive remodeling, disrupting cardiomyocyte metabolic homeostasis. As a highly energy-dependent organ, the heart relies on continuous ATP synthesis by mitochondria—ATP reserves support only limited contractions and must be replenished Via mitochondrial oxidative phosphorylation. Mitochondria also regulate contractile function and cell viability, and mitochondrial dysfunction is a hallmark of heart failure [26,27,28,29]. Prolonged pressure overload, such as in the transverse aortic constriction (TAC) model used here, impairs mitochondrial core functions: it disrupts the respiratory chain, increases electron leakage during electron transfer, and induces excessive reactive oxygen species (ROS) production [7,24]. Excessive ROS further exacerbates mitochondrial dysfunction (protein misfolding), damages cardiomyocyte structures (membranes, organelles), and activates pro-apoptotic and pro-fibrotic pathways, forming a "vicious cycle" of mitochondrial impairment, ROS accumulation, and aggravated myocardial damage [24,29,30].
In this study, TAC mice exhibited reduced left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), enlarged ventricular cavities (increased LVIDd and LVIDs), cardiomyocyte hypertrophy (HE and WGA staining), fibrosis (Masson staining), and systemic oxidative stress/metabolic disturbances (decreased serum SOD activity and ATP levels, increased serum MDA levels). Hopeaphenol treatment dose-dependently ameliorated these changes; notably, in vitro experiments confirmed HP directly improved cardiac mitochondrial function by reducing mitochondrial ROS and restoring membrane potential. These results confirm that hopeaphenol disrupts the "mitochondrial dysfunction–oxidative stress–cardiac hypertrophy" vicious cycle by improving systemic energy metabolism and augmenting antioxidant capacity.
Hopeaphenol's protective effects are closely linked to AMPK activation. AMPK, a central cellular energy sensor activated by increased AMP/ATP ratios, maintains mitochondrial homeostasis by regulating downstream targets [11,31,32]. Molecular docking confirmed that hopeaphenol interacts directly with AMPK by forming stable bonds with key active-site residues, supporting a specific molecular association between the two. CETSA experiments further validated this direct interaction, showing that hopeaphenol enhances pAMPK's thermal stability and reduces its degradation—corroborating the docking results and reinforcing that hopeaphenol binds AMPK to modulate its function. Functionally, hopeaphenol upregulated AMPK and SIRT1 expression in TAC mouse myocardium; SIRT1, a downstream AMPK target, promotes mitochondrial biogenesis and antioxidant function Via deacetylation of PGC-1α [33]. Notably, in vitro, hopeaphenol's beneficial effects on Ang II-induced HL-1 cardiomyocyte hypertrophy (reduced cell area, downregulated BNP) and mitochondrial dysfunction (decreased ROS, restored membrane potential) were abolished by the AMPK inhibitor Compound C, confirming AMPK as a critical mediator of hopeaphenol's effects.
Building on these experimental findings (direct binding Via docking/CETSA, functional upregulation of AMPK/SIRT1, and dependence on AMPK activity), we propose a model where hopeaphenol's direct binding to inactive AMPK (supported by docking and CETSA), combined with the well-established mechanism that LKB1 serves as a classical upstream phosphorylating kinase of AMPK [34], is speculated to sensitize the kinase to phosphorylation by upstream regulators like LKB1. The activation of AMPK occurs through the phosphorylation of T172, a process controlled by LKB1, and the enhanced LKB1/AMPK pathway stimulates the TSC1/TSC2 complex, subsequently suppressing mTOR activity [34]. This priming effect leads to enhanced AMPK phosphorylation (pAMPK upregulation) and subsequent activation of downstream effectors (SIRT1, PGC-1α). SIRT1 then participates in a positive feedback loop—by deacetylating PGC-1α, it promotes mitochondrial biogenesis and antioxidant function, amplifying AMPK-mediated metabolic improvements. This model explains how HP's binding initiates AMPK activation without requiring it to be a direct orthosteric activator, and highlights the potential role of upstream kinases in mediating the full signaling cascade.
This proposed model integrates our key experimental observations. These include direct hopeaphenol-AMPK binding (docking/CETSA), elevated pAMPK (WB) and SIRT1 upregulation. It also bridges a knowledge gap. It links the earlier observed hopeaphenol-AMPK binding (and subsequent stability enhancement) to the functional increase in pAMPK—resolving how physical interaction translates to pathway activation—a marker of AMPK activation and subsequent downstream activation. This link relies on speculated LKB1-mediated phosphorylation sensitization. Direct kinase assays were not performed. Even so, this framework prepares for comparing hopeaphenol with other AMPK activators like resveratrol. It highlights how hopeaphenol's unique mechanism connects to its structural and functional advantages.
While our study delineates the central role of the AMPK/SIRT1 axis, our KEGG enrichment analysis (Figure 4C) also suggests the potential involvement of other downstream pathways such as mTOR signaling and autophagy—key regulators of cardiac hypertrophy. It has been shown that the activation of AMPK/mTOR signaling is linked to the activation of autophagy [35]. Given established crosstalk between AMPK activation, mTOR inhibition (which suppresses pathological protein synthesis), and enhanced autophagic flux (which clears damaged mitochondria), these pathways may contribute to hopeaphenol's comprehensive cardioprotective effects. Due to time and scope constraints, we did not experimentally validate these pathways in the current study; future investigations will be essential to determine their specific contributions and whether they act in synergy with AMPK/SIRT1 to amplify therapeutic benefits. This potential multi-pathway regulatory feature aligns with hopeaphenol's structural basis for multi-target activity (foreshadowed in the model's emphasis on "unique mechanism"), as its multimeric structure enables interactions with multiple targets—laying the groundwork for the following discussion of structural advantages.
As a resveratrol tetramer, hopeaphenol exhibits structural and functional advantages over its monomeric parent, building on foundational work by Seya et al. [18]. Their study identified that resveratrol tetramers display divergent bioactivities: vitisin A induces cardiomyocyte apoptosis, while hopeaphenol suppresses it—underscoring that stilbene oligomer function depends on specific stereochemistry rather than just monomer composition. Our findings extend this work by demonstrating that hopeaphenol's anti-apoptotic phenotype translates to anti-hypertrophic effects Via AMPK/SIRT1 activation, providing a mechanistic explanation for its protective role first observed by Seya et al. Stilbene oligomers, including hopeaphenol, have improved biological properties: their multimeric structure enhances stability and resistance to metabolic degradation, ensuring higher bioavailability compared to resveratrol monomers. This structural feature also confers multi-target activity—hopeaphenol not only directly binds to AMPK but also interacts more effectively with molecules such as angiotensin-converting enzyme (ACE) than resveratrol [36], thereby amplifying its protective effects [18]. In contrast, resveratrol has limitations: its effects are dose-dependent and biphasic (antioxidant at low doses, pro-oxidant at ≥50 μM, causing DNA damage and mitochondrial dysfunction in endothelial cells [37,38,39,40]), with toxicity to normal cells (rat thymocytes and fibroblasts [41,42]) and potential drug–drug interactions via CYP3A4 inhibition [43,44]. In clinical settings, administering high doses (2–5 g/day) of certain compounds is often associated with gastrointestinal discomfort (such as diarrhea and nausea), abnormal liver function, and potential nephrotoxicity [45,46]. Nevertheless, our results confirm hopeaphenol's low toxicity: Hopeaphenol ameliorates cardiac hypertrophy in a dose-dependent manner at 5–20 mg/kg (in vivo) or 1–10 μM (in vitro) without significant toxicity. More importantly, hopeaphenol mediates the activation of the AMPK/SIRT1 pathway through direct binding to AMPK—this interaction is supported by comprehensive structural and functional evidence: molecular docking with two AMPK structures (cardiac-predominant α2 kinase domain, PDB 2H6D; full-length active heterotrimer, PDB 6B2E) shows hopeaphenol binds the inactive α2 isoform with higher affinity (binding energy: −9.206 kcal/mol vs. −8.083 kcal/mol for 6B2E) Via stable hydrogen bonds with GLU-143 and ASP-139; meanwhile, Cellular Thermal Shift Assay (CETSA) confirms this direct binding by significantly enhancing total AMPK thermal stability (ΔTm: +4.79 °C) compared to the DMSO control. This state-dependent, target-specific mechanism is clearer and more reliable than resveratrol, which acts independently of the AMPK pathway at low concentrations.
In a clinical context, this study identifies hopeaphenol as a promising therapeutic candidate for cardiac hypertrophy, emphasizing the significance of the AMPK/SIRT1 signaling pathway in ameliorating myocardial metabolic disorders. Given that aberrant energy metabolism is an early indicator in the progression from hypertrophy to heart failure [6], hopeaphenol's capacity to enhance cardiac function and mitigate remodeling in TAC mice indicates its potential utility for early intervention in pressure overload-induced hypertrophy (hypertension, aortic valve stenosis). However, several limitations must be addressed: (1) the study's reliance on the TAC model necessitates validation across other etiologies of hypertrophy, such as volume overload from mitral regurgitation or myocardial infarction; (2) the investigation into upstream AMPK regulators, such as LKB1, and the complete activation cascade remains incomplete; (3) Elucidating the roles of specific AMPKα subunits through genetic approaches (knockout mouse models) to confirm AMPK's essential role; (4) Developing nano-delivery systems to improve bioavailability, addressing a common challenge in translating natural polyphenols to clinical use. It is important to note that the use of Compound C, a pharmacological AMPK inhibitor, has limitations due to its potential off-target effects. While our data consistently show that Compound C blocks the beneficial effects of hopeaphenol, which strongly suggests the involvement of AMPK, this evidence alone cannot conclusively prove that the effects are exclusively mediated through AMPK. To more definitively establish the essential role of AMPK, future studies employing genetic approaches, such as siRNA-mediated knockdown or CRISPR/Cas9-mediated knockout of AMPKα in cardiomyocytes, are warranted. These methods would provide higher specificity and strengthen the conclusion that hopeaphenol's cardioprotective effects are primarily dependent on AMPK signaling.
From a translational perspective, currently, hopeaphenol holds promise as a candidate for future therapeutic development rather than an immediately translatable clinical agent. Notwithstanding the promising cardioprotective effects observed, the clinical translational potential of hopeaphenol remains to be fully established—particularly addressing the limitations of pharmacological inhibitors (Compound C) noted earlier. Future investigations are therefore imperative, with key directions including: (1) Characterizing its pharmacokinetic profile, tissue distribution, and chronic toxicity in advanced animal models; (2) Evaluating its effects on human cardiomyocyte organoids or other human-relevant systems to bridge the gap between preclinical findings and potential clinical application; (3) Elucidating the roles of specific AMPKα subunits through genetic approaches (knockout mouse models) to confirm AMPK's essential role; (4) Developing nano-delivery systems to improve bioavailability, addressing a common challenge in translating natural polyphenols to clinical use. These future directions will further validate the mechanisms underlying hopeaphenol's action.
5. Conclusions
In this study, integrated in vivo (TAC mouse model), in vitro (Ang II-induced HL-1 cardiomyocytes), and network pharmacology experiments confirm that hopeaphenol exerts a protective effect against pressure overload-induced cardiac hypertrophy and delays heart failure progression.Mechanistically, hopeaphenol directly binds to AMPK (validated by molecular docking with two AMPK structures and CETSA) to activate the AMPK/SIRT1 signaling pathway. This activation improves mitochondrial energy metabolism, reduces oxidative stress, mitigates myocardial hypertrophy and fibrosis, and ultimately restores cardiac function—effects that are abolished by the AMPK inhibitor Compound C, confirming AMPK as a critical mediator.Compared with its monomer resveratrol, hopeaphenol has structural and functional advantages: higher bioavailability (due to enhanced metabolic stability), lower toxicity (effective at 5–20 mg/kg in vivo or 1–10 μM in vitro without significant cytotoxicity), and a clearer, more target-specific mechanism (state-dependent binding to inactive AMPK, avoiding resveratrol's biphasic effects and off-target risks).
Collectively, these findings identify hopeaphenol as a promising candidate for the prevention and treatment of cardiac hypertrophy-related heart failure, while also highlighting the need for further research: validating its efficacy in diverse hypertrophy models, clarifying the role of specific AMPKα subunits via genetic approaches, and optimizing nano-delivery systems to improve its clinical translational potential (Figure 8).