Aurantiamide Acetate Mitigates Hypertensive Cardiac Hypertrophy via Modulation of MAPK Signaling and ER Stress

Aurantiamide Acetate Mitigates Hypertensive Cardiac Hypertrophy via Modulation of MAPK Signaling and ER Stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Aurantiamide Acetate Mitigates Hypertensive Cardiac Hypertrophy via Modulation of MAPK Signaling and ER Stress Tuğçe Çeker, Mutay Aslan, Esma Kırımlıoğlu, Hazal Tuzcu Balaban, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6939373/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Purpose Hypertensive cardiac hypertrophy contributes significantly to heart failure and is mediated by maladaptive mechanisms including mitogen-activated protein kinase (MAPK) signaling and endoplasmic reticulum (ER) stress. Aurantiamide acetate (AA), a dipeptide derivative derived from edible plants and marine fungi, exhibits anti-inflammatory and vasoprotective effects. However, its impact on hypertensive cardiac hypertrophy remains unclear. This study aims to explore the therapeutic potential of AA in mitigating cardiac hypertrophy by targeting MAPK signaling and ER stress pathways. Methods Cellular hypertrophy was induced in H9C2 cardiomyoblasts using angiotensin II (Ang II), and tunicamycin (TM) was used to trigger ER stress. AA’s effects were assessed via viability assays, immunofluorescence staining, and ELISA for hypertrophic and ER stress markers. In vivo, hypertensive hypertrophic cardiomyopathy (HCM) was established in rats using a two-kidney, one-clip (2K1C) model, followed by AA administration. Cardiac hypertrophy, fibrosis, MAPK activation, and ER stress markers were evaluated histologically and biochemically. Results In vitro, AA significantly reduced Ang II-induced cardiomyocyte hypertrophy and ER stress, as evidenced by decreased cell size, atrial natriuretic peptide (ANP) expression, and glucose-regulated protein 78 (GRP78) levels. In vivo, AA ameliorated hypertension-induced cardiac remodelling, reduced myocardial fibrosis, suppressed MAPK phosphorylation (p38, extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK]), and alleviated ER stress. Blood pressure and serum Ang II levels were also significantly lowered in AA-treated rats. Conclusion Aurantiamide acetate mitigates hypertensive cardiac hypertrophy by inhibiting MAPK activation and ER stress both in vitro and in vivo, highlighting its potential as a novel therapeutic agent for hypertensive heart disease. Aurantiamide acetate Hypertensive cardiac hypertrophy MAPK signaling Endoplasmic reticulum stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Points • AA reduced cardiomyocyte hypertrophy and ER stress in H9C2 cells. • AA suppressed MAPK pathway activation in vitro and in vivo. • AA decreased myocardial fibrosis and improved cardiac remodelling. • AA lowered blood pressure and serum Ang II levels in hypertensive rats. Introduction Hypertrophic cardiomyopathy is a primary myocardial disorder characterized by excessive thickening of the myocardium, most notably the left ventricle, in the absence of abnormal loading conditions such as hypertension or valvular disease [ 1 ]. However, hypertensive-induced cardiac hypertrophy shares pathological similarities with HCM, including myocardial fibrosis, cardiomyocyte hypertrophy, and adverse ventricular remodelling, ultimately predisposing individuals to heart failure, arrhythmias, and sudden cardiac death [ 2 ]. Understanding the molecular mechanisms underlying hypertrophy in hypertension is critical for developing targeted therapies to prevent or reverse disease progression. Angiotensin II plays a pivotal role in hypertensive cardiac remodelling by promoting cardiomyocyte growth, fibrosis, and inflammation through activation of the renin-angiotensin-aldosterone system (RAAS) [ 3 ]. Binding of Ang II to the angiotensin type 1 receptor (AT1R) initiates multiple intracellular signaling cascades, among which the MAPK pathway, including p38 MAPK, ERK, and JNK, is heavily implicated in mediating hypertrophic responses [ 4 ]. In addition to promoting hypertrophic growth, MAPK signalling has been linked to the induction of ER stress, a condition resulting from the accumulation of misfolded proteins that triggers the unfolded protein response (UPR) [ 5 ]. Persistent ER stress exacerbates cardiac dysfunction by impairing cellular homeostasis and promoting apoptosis [ 6 ]. Recent studies have illuminated the intersection between MAPK activation and ER stress in driving cardiac hypertrophy, suggesting that agents targeting these pathways could offer therapeutic benefits [ 7 , 8 ]. Despite this knowledge, clinically effective interventions that modulate these pathways remain limited, underscoring the need for novel therapeutic strategies. Aurantiamide acetate, a dipeptide derivative commonly found in edible plants such as Portulaca oleracea L. (purslane) and marine-derived fungi ( Aspergillus species), has attracted attention for its diverse pharmacological properties, including anti-inflammatory, antioxidant, and anti-cancer activities [ 9 ]. Structurally, AA consists of N-benzoylphenylalanine and phenylalanine residues, conferring it the ability to modulate key biological pathways [ 10 ]. Previous investigations have demonstrated that AA exerts vasoprotective effects by enhancing endothelial nitric oxide synthase (eNOS) expression, improving vascular reactivity, and reducing oxidative stress in experimental models of hypertension [ 11 ]. In particular, AA was shown to attenuate mean blood pressure, improve endothelial-dependent relaxation, and enhance red blood cell deformability in rats subjected to 2K1C renovascular hypertension, suggesting a multifaceted role in cardiovascular protection [ 11 ]. Despite the growing body of research highlighting the pathological roles of MAPK signaling and ER stress in cardiac hypertrophy [ 5 ], and the known beneficial effects of AA on vascular function, the potential cardioprotective role of AA in hypertensive HCM had not been previously explored [ 11 ]. Importantly, while AA's vascular effects were studied in hypertensive models, its direct impact on cardiomyocytes, particularly in the context of hypertrophic growth and ER stress modulation, remained unclear. Furthermore, there was a lack of comprehensive investigations integrating both in vitro and in vivo approaches to elucidate the molecular mechanisms underlying AA’s potential cardioprotective actions. To address these gaps, the present study was designed to evaluate the effects of AA on hypertensive cardiac hypertrophy, focusing on its ability to modulate MAPK signaling and ER stress. The study employed a two-pronged approach: first, using H9C2 cardiomyoblasts to examine the direct effects of AA on cellular hypertrophy and ER stress markers in vitro; and second, employing a 2K1C rat model to validate the in vivo cardioprotective effects of AA against hypertension-induced hypertrophic remodelling. This integrated strategy aimed to provide novel insights into the therapeutic potential of AA as a modulator of hypertrophic signaling pathways and ER stress, offering a promising avenue for the treatment of hypertensive heart disease. Therefore, we hypothesized that AA could attenuate cardiac hypertrophy by inhibiting MAPK signaling and alleviating ER stress. The primary objective of this study was to evaluate the effects of AA on hypertrophic remodelling and ER stress responses in Ang II-stimulated H9C2 cardiomyoblasts. A secondary objective was to validate these findings in vivo using a 2K1C rat model of hypertensive HCM by assessing myocardial structure, fibrosis, hypertrophy markers, and ER stress-related protein expression. Through this integrated approach, we aimed to provide new mechanistic insights into the cardioprotective potential of AA and its relevance as a therapeutic candidate for hypertensive cardiac disease. Materials and Methods Cell Culture The rat cardiomyoblast cell line, H9C2 (ATCC® CRL-1446™) was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained in low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; D6046, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 1000 mg/L L-glutamine and sodium bicarbonate. The medium was further enriched with 10% (v/v) fetal bovine serum (FBS; Capricorn Scientific, FBS-11A, Ebsdorfergrund, Germany), 1% (v/v) penicillin (100 U/mL) and streptomycin (100 µg/mL) (Gibco, Life Technologies Corporation, 15140-122, Grand Island, NY, USA), and Amphotericin-B at a concentration of 200 µL/L (Gibco, Life Technologies Limited, 15290-026, Paisley, UK). The complete medium was sterilized using a 0.22 µm bottle-top filter and stored at 4°C until use. Cells were cultured at 37°C in a humidified incubator with 5% CO₂ and 95% air. Upon reaching approximately 80% confluence, cells were subcultured using 0.05% trypsin-EDTA (1X) (Gibco, Life Technologies Corporation, 25300054, Grand Island, NY, USA). Induction of Cellular Hypertrophy Cellular hypertrophy was induced using Ang II (Sigma-Aldrich, A9525, Darmstadt, Germany) [ 12 ]. The powdered compound was dissolved in sterile distilled water to prepare a 24 mM stock solution. Working solutions at final concentrations of 0.01, 0.1, 0.2, 1, and 10 µM were obtained by diluting the stock solution with cell culture medium and applied to the cells for 24 hours. To verify that the hypertrophic effects of Ang II were mediated via the angiotensin II type 1 receptor (AT1R), the selective AT1R antagonist losartan (Los; Sigma-Aldrich, SML3317, Darmstadt, Germany) was used as a negative control [ 13 ]. Losartan was dissolved in dimethyl sulfoxide (DMSO; Fisher Scientific, BP231-1, Leicestershire, UK) to prepare a 10 mM stock solution. Final concentrations of 5 µM and 10 µM were administered to the cells 1 hour prior to Ang II treatment. Aurantiamide Acetate Treatment Aurantiamide acetate (MedChemExpress, HY-N2905, Monmouth Junction, NJ, USA) was obtained in powder form and dissolved in DMSO to prepare a 100 mM stock solution. The stock was subsequently diluted with culture medium to achieve final concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 µM. These concentrations were applied to the cells 24 hours [ 11 ]. Induction and Inhibition of Endoplasmic Reticulum Stress Endoplasmic reticulum stress was induced using TM (Abcam, ab120296, Cambridge, UK) [ 14 ]. TM was dissolved in DMSO to prepare a 10 mg/mL stock solution. This stock was diluted in cell culture medium to final concentrations of 0.5, 1, 2, 5, and 10 µg/mL and applied to the cells for 24 hours. Tauroursodeoxycholic acid (TUDCA; Millipore, 580549, Darmstadt, Germany), a known ER stress inhibitor, was used to attenuate TM-induced stress [ 14 ]. TUDCA was dissolved in sterile 0.9% isotonic sodium chloride (NaCl) to prepare a 10 mM stock solution. Final concentrations of 0.06, 0.125, 0.5, 1, 2, and 4 mM were administered to the cells 12 hours after the initiation of TM treatment. Cell Viability Assay Cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; GoldBio, T-030-1, St. Louis, MO, USA) colorimetric assay. MTT powder was dissolved in 1X phosphate-buffered saline (PBS; Sigma-Aldrich, P4417, St. Louis, MO, USA) at a concentration of 5 mg/mL, following the manufacturer’s protocol, and sterilized by filtration through a 0.22 µm membrane filter. H9C2 cells were seeded at a density of 5,000 cells per well in 96-well plates and incubated for 24 hours to allow adherence. Subsequently, cells were treated with Ang II, Los, AA, TM, or TUDCA at the designated concentrations and exposure times, in a final volume of 200 µL per well. At the end of the treatment period, MTT solution was added to each well to achieve a final concentration of 0.5 mg/mL. Plates were then incubated for 2 hours at 37°C in a humidified atmosphere containing 5% CO₂, protected from light. Following incubation, the culture medium was aspirated, and the resulting formazan crystals were dissolved in 100 µL of DMSO. Absorbance was recorded using a spectrophotometer at 570 nm, with background subtraction at 690 nm. Cell viability was expressed as a percentage relative to the untreated control group, calculated by the formula: Cell viability (%) = (Abs_sample / Abs_control) × 100. Based on MTT cell viability assay results, non-cytotoxic and experimentally appropriate treatment groups were established as follows: Control cells were cultured in low-glucose DMEM alone; the DMSO group received 1 µL/mL DMSO (vehicle control) for 24 hours; hypertrophy was induced by treating cells with 1 µM Ang II for 24 hours (Ang II group); cells treated with 6.25 µM AA for 24 hours constituted the AA group, while the Ang II + AA group received combined treatment for 24 hours. The Los group was treated with 10 µM for 25 hours; in the Los + Ang II group, Los was administered 1 hour prior to 24-hour Ang II exposure, and in the Los + Ang II + AA group, Los pretreatment was followed by co-administration of Ang II and AA for 24 hours. ER stress was induced by treating cells with 0.5 µg/mL TM for 24 hours (TM group). Co-treatment groups included TM + Ang II, TM + AA, and TM + Ang II + AA, all for 24 hours. Additionally, the TM + TUDCA group involved a total 24-hour incubation where 1 mM TUDCA was added 12 hours post-TM treatment, while in the TM + Ang II + TUDCA group, TUDCA was administered 12 hours after combined TM and Ang II treatment. Cell Surface Area Measurement Cell surface area was quantified using a fluorescent phalloidin conjugate specific for F-actin filaments (Phalloidin-iFluor™ 555 Conjugate; Santa Cruz Biotechnology, sc-363794, Dallas, TX, USA) [ 15 ]. The lyophilized reagent was reconstituted in DMSO to prepare a 1000X stock solution, which was stored at − 20°C until use. H9C2 cells were seeded at a density of 50,000 cells per well into 8-well chamber slides (Merck Millipore, PEZGS0816, Cork, Ireland) and incubated overnight to ensure adherence. The following day, after confirming 70–80% confluency and normal morphology, cells were treated with experimental media according to group-specific concentrations and incubation times. Following treatment, cells were washed twice with cold PBS and fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich, 158127, St. Louis, MO, USA) for 10 minutes. After three PBS washes, cell membranes were permeabilized using 0.2% Triton X-100 (Sigma-Aldrich, T8787) for 10 minutes, followed by five additional PBS washes. A 1X staining solution was prepared by diluting the phalloidin stock 1:1000 in PBS containing 1% BSA (Sigma-Aldrich, A8806) and incubated with cells at room temperature for 45 minutes. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, H-1200) and mounted with coverslips, ensuring the absence of air bubbles. Fluorescence images were acquired using an Olympus IX81 inverted microscope (Olympus Corporation, Tokyo, Japan) equipped with TRITC and DAPI filter sets at 20× magnification. Image analysis was performed using ImageJ software (version 1.54m; NIH, Bethesda, MD, USA). Images were converted to RGB stack format, and thresholding was applied to isolate cell regions. Using the ROI Manager tool, cell boundaries were manually delineated based on F-actin signals, and the surface area (µm²) of at least 20 individual cells per group was measured. Data was subjected to statistical analysis to evaluate differences between experimental groups. Immunofluorescence Staining To assess the expression of target proteins in H9C2 cells, immunofluorescence staining was performed. Cells at approximately 80% confluency were detached using 0.05% trypsin-EDTA and seeded onto 8-well chamber slides at a density of 20,000 cells per well. After overnight incubation to allow cell adhesion, confluency (70–80%) and normal morphology were confirmed microscopically. Cells were then treated with the designated media corresponding to each experimental group. At the end of the treatment period, culture media were removed, and cells were washed twice with PBS. Fixation was performed with 4% paraformaldehyde for 10 minutes at room temperature, followed by three PBS washes. Cells were permeabilized using 0.2% Triton X-100 in PBS for 10 minutes and then washed five times with PBS. To block nonspecific antibody binding, cells were incubated with 5% normal goat serum (NGS; Vector Laboratories Inc., S-1000-20; Newark, CA, USA) in PBS for 30 minutes at room temperature. Subsequently, cells were incubated overnight at 4°C in the dark with 200 µL per well of the following primary antibodies, each diluted 1:200 in PBS containing 1% BSA: rabbit anti-GRP78 (Abcam, ab21685; Cambridge, MA, USA), anti-p38 MAPK (Affinity Biosciences, AF6456; Cincinnati, OH, USA), anti-phospho-p38 MAPK (Thr180/Tyr182; Affinity Biosciences, AF4001), anti-ERK1/2 (Affinity Biosciences, AF0155), anti-phospho-ERK1/2 (Thr202/Tyr204; Affinity Biosciences, AF1015), anti-JNK1/2/3 (Affinity Biosciences, AF6319), and anti-phospho-JNK1/2/3 (Thr183 + Tyr185; Affinity Biosciences, AF3318). On the following day, cells were washed five times with PBS and incubated for 45 minutes at room temperature in the dark with a goat anti-rabbit IgG secondary antibody conjugated to Alexa Fluor® 488 (1:1000; Abcam, ab150077; Cambridge, UK) and F-actin counterstain using Phalloidin-iFluor™ 555 (1X; Abcam, ab176756; Cambridge, UK), both diluted in PBS with 1% BSA at a volume of 200 µL per well. After three additional PBS washes, the chamber structure was removed, and nuclei were counterstained with DAPI (Vector Laboratories, H-1200; Newark, CA, USA) by adding a drop to each well and mounting coverslips without air bubbles. Fluorescence images were acquired using an Olympus IX81 inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with FITC (Alexa 488), TRITC (Phalloidin 555), and DAPI filter sets, at 20× magnification. Image analysis was conducted using ImageJ software (version 1.54m; NIH, Bethesda, MD, USA). Fluorescently labeled cell regions were manually delineated for each group, and fluorescence intensity was quantified using the Integrated Density function. Corrected Total Cell Fluorescence (CTCF) was calculated using the formula: CTCF = Integrated Density − (Area of selected cell × Mean background fluorescence). In Vivo Model of Cardiac Hypertrophy All animal procedures were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee of the Akdeniz University Experimental Animal Research Center (Ethics Committee Approval No: 6; Date: 05.02.2024; Protocol No: 1677/2024.02.003). One-month-old male albino Wistar rats (180–240 g) were housed in stainless steel cages (four rats per cage) under controlled environmental conditions: 20–24°C ambient temperature, 40–60% relative humidity, and a 12-hour light/dark cycle. Animals had free access to standard rodent chow and water throughout the study. Rats were randomly assigned to one of five groups (n = 8 per group): (1) Control, (2) Sham-operated (Sham), (3) AA (4) Hypertensive HCM model (HCM), and (5) HCM treated with AA (HCM + AA). The HCM model was induced using the two-kidney, one-clip (2K1C) method, a well-established technique that promotes cardiac remodelling through activation of the renin–angiotensin–aldosterone system (RAAS) [ 16 ]. The procedure was performed as previously described [ 11 ]. Briefly, rats were anesthetized via intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). Following shaving and disinfection of the abdominal area with povidone-iodine, a midline laparotomy was performed. The left kidney was gently exteriorized, and the left renal artery was carefully isolated. A non-absorbable 5/0 silk suture (Boz Tıbbi Malzemeler San. Tic. A.Ş., Ankara, Turkey), coated with silicone, was placed around the renal artery using a 0.22 mm diameter guide to achieve partial occlusion. In the Sham group, the same surgical steps were performed without arterial constriction. Upon completion of the surgical procedure, the peritoneum was sutured with a 3/0 absorbable multifilament glycolic acid suture, and the skin was closed using a non-absorbable 5/0 silk suture coated with silicone (Boz Tıbbi Malzemeler San. Tic. A.Ş., Ankara, Turkey). To verify the establishment of hypertension in the 2K1C-induced groups, systolic and diastolic blood pressures (DBP) were measured weekly using a non-invasive tail-cuff system (MAY-BPHR 9610-PC; BIOPAC Systems, Santa Barbara, CA, USA). For each rat, three consecutive readings were obtained, and the average values were calculated. Measurements were conducted between 09:00 and 12:00 to minimize the influence of circadian variations. The hypertensive state was monitored over a 4-week period to confirm successful model induction. Aurantiamide Acetate Administration Protocol A stock solution of AA was prepared at a concentration of 100 mM by dissolving 10 mg of the compound in 225 µL of DMSO. For each rat, 2.5 µL of this stock solution—corresponding to the appropriate dose for a 200 g animal—was diluted in DMSO to a final volume of 100 µL and administered via intraperitoneal (i.p.) injection. The treatment regimen consisted of daily administration at a dose of 0.5 mg/kg, five days per week (totaling 2.5 mg/kg/week), for four consecutive weeks. The total cumulative dose of AA administered per animal over the treatment period was 10 mg/kg. To control for potential vehicle effects, animals in the control, sham-operated, and HCM-only groups received equivalent volumes (100 µL) of DMSO via i.p. injection following the same dosing schedule. At the conclusion of the four-week treatment period, animals were anesthetized with an intraperitoneal injection of ketamine (45 mg/kg) and xylazine (10 mg/kg), and subsequently euthanized. Hearts were rapidly excised. For histological evaluations, longitudinal cardiac sections were collected from four animals per group and fixed in 10% neutral-buffered formalin for hematoxylin-eosin (H&E), Masson’s trichrome, and immunohistochemical staining. For biochemical analyses, additional cardiac tissues were promptly snap-frozen in liquid nitrogen and stored for subsequent processing. Evaluation of Cardiac Hypertrophy by Hematoxylin and Eosin Staining Cardiac tissues were fixed, embedded in paraffin, and sectioned at a thickness of 4–5 µm using a microtome. To assess morphological alterations indicative of cardiac hypertrophy, the tissue sections were stained with H&E following standard histological protocols [ 17 ]. Stained sections were examined under a Zeiss Primo Star light microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany) at 40× magnification. High-resolution images were captured, and the cross-sectional area (CSA) of individual cardiomyocytes was quantified using Fiji software (ImageJ, version 1.54p; National Institutes of Health, Bethesda, MD, USA). Cell boundaries were manually delineated, and the average cardiomyocyte surface area (µm²) was calculated from multiple fields per sample to ensure representative measurements. Quantitative Assessment of Myocardial Fibrosis Using Masson’s Trichrome Staining Masson’s Trichrome staining was employed to evaluate fibrotic changes in the left ventricular myocardium [ 18 ]. Fixed heart tissues were embedded in paraffin and sectioned at a thickness of 5 µm. The sections were deparaffinized and rehydrated through a graded series of xylene and ethanol solutions. Staining was performed manually according to established protocols described in the literature [ 19 ]. In the stained sections, muscle fibers appeared purple, collagen fibers blue-green, and nuclei were stained dark blue to black. Representative fields from each experimental group were randomly selected, and images were captured at 40× magnification using a Zeiss Primo Star light microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany). Fibrosis was quantified by calculating the collagen volume fraction (CVF). Image analysis was conducted using Fiji software (ImageJ version 1.54p; National Institutes of Health, Bethesda, MD, USA). The images were subjected to color deconvolution using the “Color → Color Deconvolution → Masson Trichrome” function to separate staining channels. Thresholding was applied specifically to the aniline blue channel, corresponding to collagen deposition. The percentage of the collagen-positive area was then calculated, and CVF was determined using the following formula: CVF (%) = (Collagen Area / Total Tissue Area) × 100. Immunohistochemical Staining of Cardiac Tissue Longitudinal sections of cardiac tissue were used for histological assessment and immunohistochemical analysis of target protein expression. Following fixation, the tissues were rinsed with distilled water, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Serial sections of 4 µm thickness were obtained using a microtome and mounted onto poly-L-lysine-coated glass slides. The sections were incubated overnight at 45°C, followed by an additional hour at 60°C to ensure adhesion and optimal antigen preservation prior to staining. For deparaffinization, slides were immersed in xylene twice for 20 minutes each. Rehydration was subsequently performed through descending concentrations of ethanol (100%, 90%, 80%, and 70%) for 5 minutes per step. Slides were then rinsed in distilled water and PBS (pH 7.4) for 5 minutes each. Immunohistochemical staining was conducted using the Dako Omnis automated staining system (Agilent Technologies, Santa Clara, CA, USA). Primary antibodies were diluted in PBS containing 1% bovine serum albumin (BSA) and applied at room temperature (25°C) for 60 minutes. The following primary antibodies were used: Polyclonal rabbit anti-GRP78 (1:100; Abcam, ab21685), Anti-p38 MAPK (1:50; Affinity Biosciences, AF6456), Anti-phospho-p38 MAPK (Thr180/Tyr182) (1:50; Affinity Biosciences, AF4001), Anti-ERK1/2 (1:100; Affinity Biosciences, AF0155), Anti-phospho-ERK1/2 (Thr202/Tyr204) (1:50; Affinity Biosciences, AF1015), Anti-JNK1/2/3 (1:50; Affinity Biosciences, AF6319), Anti-phospho-JNK1/2/3 (Thr183 + Tyr185) (1:100; Affinity Biosciences, AF3318), Secondary detection was performed using biotinylated goat anti-rabbit IgG, followed by streptavidin-conjugated peroxidase. Immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB) substrate, resulting in a brown precipitate indicative of positive staining. Negative controls were processed using the same protocol, substituting 5% NGS for the primary antibody. Microscopic imaging was performed using a Zeiss Primo Star light microscope at 40× magnification. Quantitative analysis of immunostaining was carried out using Fiji (ImageJ, version 1.54p). Color deconvolution was applied via the "Color → Color Deconvolution → H-DAB" function to isolate the DAB channel. A consistent threshold for positive signal detection was manually set and applied across all images. The mean gray value of positively stained regions was measured, and optical density (OD) was calculated using the following formula: OD = log₁₀(255 / mean gray value). OD values from the control group were designated as 100% to represent baseline staining intensity, and all experimental groups were normalized accordingly as percentage values relative to the control. All data were subjected to statistical analysis. Enzyme-Linked Immunosorbent Assay (ELISA) Serum concentrations of Ang II were quantified using a commercially available sandwich ELISA kit (BT-Laboratory, Cat. No: E0655Ra, Zhejiang, China), following the manufacturer's protocol. Diluted serum samples were applied to microplate wells pre-coated with anti-Ang II antibodies. After incubation, streptavidin-conjugated horseradish peroxidase (HRP) was added, followed by a chromogenic substrate to facilitate colour development. The enzymatic reaction was terminated using a stop solution, and absorbance was measured at 450 nm using a microplate spectrophotometer. A standard calibration curve was generated for each assay, and Ang II concentrations were calculated based on this curve. Final results were expressed in pmol/L. The expression levels of ANP, GRP78, p38 MAPK, ERK, and JNK were determined in whole heart tissue homogenates and H9C2 cell lysates using specific sandwich ELISA kits (BT-Laboratory, Zhejiang, China; ANP: Cat. No. E0642Ra; GRP78: E1255Ra; p38: E2455Ra; ERK: E1090Ra; JNK: E0048Ra). For tissue preparation, heart samples were rinsed with cold PBS and homogenized in PBS at a 1:9 (w/v) ratio. Homogenates were centrifuged at 5000 × g for 5 minutes at 4°C, and the supernatants were collected for analysis. H9C2 cells were harvested according to experimental group, washed with PBS, and lysed by repeated freeze-thaw cycles followed by sonication. Each assay was performed in accordance with the respective manufacturer’s instructions. Samples were added to wells precoated with specific capture antibodies and incubated with biotinylated detection antibodies. Following the application of streptavidin-HRP and chromogenic substrate, absorbance was measured at 450 nm. Standard curves were constructed for each protein, and corresponding concentrations were extrapolated accordingly. Total protein concentrations in each sample were measured using a standard protein assay, and ELISA results were normalized to the total protein content. Protein Quantification Total protein concentrations in whole heart tissue homogenates and H9C2 cell lysates were determined using a Coomassie Plus Protein Assay Kit (Pierce Chemical Company, Rockford, IL, USA), with absorbance measured at 595 nm. A standard calibration curve was generated using serial dilutions of BSA, and sample protein concentrations were calculated based on this curve. Statistical Analysis All statistical analyses were conducted using GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, CA, USA). The specific statistical tests applied for each dataset are detailed in the corresponding figure legends. Data distributions were first assessed for normality. For datasets that did not meet the assumption of normality, appropriate non-parametric tests were utilized. Comparisons among multiple groups were performed using either one-way analysis of variance (ANOVA) or the Kruskal–Wallis test, depending on data distribution. When overall significance was detected, appropriate post hoc tests were applied to determine pairwise differences between groups. A p-value of less than 0.05 was considered statistically significant. RESULTS 1. Assessment of Cell Viability and Cellular Cardiac Hypertrophy The effects of Ang II, AA, and LOS on cell viability and cardiac hypertrophy in H9C2 cells were evaluated using the MTT assay, F-actin staining, and measurements of ANP and total protein levels (Fig. 1 ). A dose-dependent cytotoxicity analysis of Ang II (0.01, 0.1, 0.2, 1, and 10 µM) was conducted after 24 hours of incubation using the MTT assay. No statistically significant differences in cell viability were observed among the groups (p = 0.3034) (Fig. 1 A). To determine the cytotoxic potential of AA, cells were treated with increasing concentrations (3.125–100 µM) for 24 hours. While no cytotoxicity was detected at low to moderate doses, the highest concentration (100 µM) significantly reduced cell viability compared to all other doses (p < 0.05) (Fig. 1 B). The cytotoxic effects of Los were assessed at 5 and 10 µM following a 25-hour incubation. No significant differences in viability were observed between these groups (p = 0.2392) (Fig. 1 C). To evaluate potential interactions, DMSO (1 µL/mL), Ang II (1 µM), AA (6.25 µM), and Los (10 µM) were administered individually or in combination. No significant differences in cell viability were observed among the groups (p = 0.1911) (Fig. 1 D). These results indicate that the selected concentrations of these agents did not exhibit cytotoxic effects. To assess morphological changes associated with hypertrophy, F-actin was stained with phalloidin (Fig. 1 E). Control cells displayed normal morphology with well-defined borders, whereas Ang II-treated cells exhibited marked enlargement. Treatment with AA and/or Los visually attenuated the Ang II-induced morphological changes. These observations were corroborated by quantitative cell surface area analysis. Compared to controls, Ang II significantly increased cell surface area (p < 0.001), which was markedly reduced by AA and Los treatments. Notably, in the Ang II + AA and Los + Ang II + AA groups, surface area values approached those of the control group (Fig. 1 F). ANP protein levels, a molecular marker of cardiac hypertrophy, were measured in cell lysates using ELISA. ANP expression was significantly elevated in the Ang II group, whereas co-treatment with losartan, AA, and Ang II significantly reduced ANP levels (p < 0.05) (Fig. 1 G). Analysis of total protein content revealed the highest levels in the Ang II-treated group. This increase was significantly attenuated by AA, LOS, and their combination therapies (p < 0.001) (Fig. 1 H). 2. Effects of Endoplasmic Reticulum Stress on Cellular Cardiac Hypertrophy The involvement of ER stress in cardiac hypertrophy and the efficacy of therapeutic interventions targeting this stress were investigated in H9C2 cells. Key parameters assessed included cell viability, GRP78 expression levels, F-actin staining for morphological analysis, and total protein content (Fig. 2 ). A model of ER stress was established using TM, and the modulatory effects of TUDCA and AA were systematically evaluated. The cytotoxic effects of TM were assessed via MTT assay following 24-hour incubation across a concentration range of 0.5–10 µg/mL. TM treatment at 1, 2, 5, and 10 µg/mL significantly reduced cell viability compared to the control group (p < 0.05), while 0.5 µg/mL did not result in a statistically significant cytotoxic effect (Fig. 2 A). TUDCA, a known ER stress inhibitor, was evaluated at doses ranging from 0.06–4 mM over 24 hours. While low to moderate concentrations were well tolerated, the highest dose (4 mM) significantly decreased cell viability (p < 0.05). Based on these findings, 1 mM TUDCA—identified as the highest non-cytotoxic concentration—was selected for subsequent experiments (Fig. 2 B). To examine the combined effects of various agents, DMSO (1 µL/mL), TM (0.5 µg/mL), Ang II (1 µM), TUDCA (1 mM), and AA (6.25 µM) were applied individually and in combination. A significant reduction in cell viability was observed only in the group receiving TM, Ang II, and AA concurrently, with viability declining to approximately 74% (p < 0.05). No significant viability changes were noted in the other treatment groups (Fig. 2 C). GRP78, a molecular marker of ER stress, was assessed using both immunofluorescence (IF) staining and ELISA (Figs. 2 D–F). IF analysis revealed that TM and Ang II significantly elevated GRP78 expression, with the TM + Ang II group showing the most pronounced increase (p < 0.001). Co-treatment with either TUDCA or AA markedly attenuated this elevation (p < 0.001) (Fig. 2 E). ELISA data obtained from cell lysates corroborated the IF results (Fig. 2 F). Morphological changes associated with hypertrophy were visualized via phalloidin-based F-actin staining (Fig. 2 G). Quantitative analysis demonstrated a significant reduction in cell surface area in the TM-treated group (p < 0.05), whereas no significant morphological alterations were observed in the other groups (Fig. 2 H). Total protein analysis showed significantly elevated levels in both TM and TM + Ang II groups compared to control cells (p < 0.01). This increase was significantly suppressed by TUDCA and AA co-treatments (TM + TUDCA, TM + AA) (p < 0.001). Notably, protein levels in the TM + Ang II + AA group were further reduced compared to those in the TM + Ang II + TUDCA group (p < 0.001) (Fig. 2 I). Collectively, these findings indicate that ER stress-induced protein accumulation in H9C2 cells can be mitigated by both AA and TUDCA, with AA demonstrating a potentially greater protective effect. These results support the therapeutic potential of AA in conditions characterized by ER stress-associated cardiac hypertrophy. 3. In Vivo Evaluation of Ang II and Endoplasmic Reticulum Stress in Hypertensive Hypertrophic Cardiomyopathy 3.1. Blood Pressure Changes and Ang II Levels In a HCM model, changes in systolic and DBP, as well as serum Ang II levels, were assessed over a 4-week period (Fig. 3 A–C). Baseline systolic blood pressure (SBP) was comparable across all groups (p > 0.05), with no significant difference observed between the control and sham groups throughout the study (p > 0.05). In the HCM group, SBP increased significantly from the first week (*, p < 0.01). A similar increase was observed in the HCM + AA group during the first week (*, p < 0.01); however, SBP gradually declined in subsequent weeks and returned to baseline by week four. SBP in the HCM group was significantly higher than in the control, sham, and AA groups at all time points (#, p < 0.05). In the HCM + AA group, SBP was elevated during the first three weeks compared to the control groups (#, p 0.05). From the second week onward, SBP in the HCM + AA group was significantly lower than in the untreated HCM group (§, p < 0.001). Additionally, SBP in the AA group was significantly lower than in the control at week two (¶, p 0.05), with no significant differences between control and sham groups during the study period (p > 0.05). DBP in the HCM group increased significantly from the first week onward (*, p < 0.05), and a similar pattern was observed in the HCM + AA group during the first week (*, p < 0.05). However, DBP declined in later weeks and returned to baseline by week four. DBP was significantly higher in the HCM group compared to control, sham, and AA groups at all time points (#, p < 0.01). In the HCM + AA group, DBP was higher during the first three weeks (#, p 0.05). Notably, DBP in the HCM + AA group decreased significantly from the second week, with values lower than those in the HCM group (§, p < 0.001). At week three, DBP in the AA group was significantly lower than both the HCM + AA and control groups (¶, p < 0.05) (Fig. 3 B). Serum Ang II levels, assessed by ELISA at the end of week four, were significantly elevated in the HCM group compared to all other groups (p < 0.001), indicating systemic activation of the RAAS in this renovascular hypertension model (Fig. 3 C). AA treatment markedly reduced this elevation in the HCM + AA group. 3.2. Modeling Hypertrophic Cardiomyopathy In Vivo Histological and biochemical assessments were performed on left ventricular myocardium to evaluate hypertrophic remodelling (Fig. 3 D–G). H&E staining of longitudinal heart sections revealed a marked increase in cardiomyocyte size in the HCM group (Fig. 3 D), which was confirmed quantitatively by cross-sectional area analysis. Average cardiomyocyte cross-sectional area in the HCM group was significantly higher than in all other groups (p < 0.001), and AA treatment significantly reduced this hypertrophic response (Fig. 3 E). ANP, a molecular marker of cardiac hypertrophy, was assessed by ELISA. ANP levels were significantly elevated in the HCM group compared to all other groups (p < 0.05), whereas AA treatment reduced ANP expression to near-control levels (p < 0.05) (Fig. 3 F). Similarly, total cardiac protein concentration was significantly elevated in the HCM group (p < 0.05), while AA treatment suppressed this increase (Fig. 3 G). 3.3. Fibrosis Analysis Cardiac fibrosis was assessed by Masson's trichrome staining of left ventricular myocardium (Fig. 3 H–I). Prominent collagen accumulation was observed in the HCM group (black arrows), while control, sham, and AA groups exhibited a more organized myocardial structure with minimal fibrotic areas. In the HCM + AA group, fibrosis was markedly reduced, and tissue architecture appeared more preserved (Fig. 3 H). Collagen volume fraction quantified using ImageJ, was significantly higher in the HCM group (50.07 ± 6.26%) compared to control (18.54 ± 6.93%), sham (16.44 ± 8.71%), AA (19.57 ± 4.23%), and HCM + AA (25.48 ± 4.04%) groups (p < 0.001). AA treatment significantly attenuated collagen accumulation (p < 0.001) (Fig. 3 I). 3.4. Cardiac Hypertrophy Assessment Absolute heart weight and heart weight-to-body weight ratio (HW/BW) were measured to assess cardiac remodelling. Heart weight was significantly higher in the HCM group (936.5 ± 39.00 mg) than in the control (734.8 ± 9.64 mg), sham (738.0 ± 37.71 mg), AA (685.3 ± 23.84 mg), and HCM + AA (802.0 ± 32.02 mg) groups (p < 0.0001). AA treatment significantly suppressed the HCM-induced increase in heart weight (p < 0.05) ( Fig. 3 J). Similarly, HW/BW ratio was significantly elevated in the HCM group (3.725 ± 0.379) compared to control (2.897 ± 0.449), sham (2.797 ± 0.130), and AA (2.854 ± 0.340) groups (p < 0.05), and was significantly reduced in the HCM + AA group (p < 0.05) (Fig. 3 K). These findings demonstrate that AA mitigates cardiac hypertrophy and relative cardiac loading associated with hypertensive remodelling. 3.5. In Vivo Assessment of Endoplasmic Reticulum Stress To evaluate ER stress in hypertensive HCM, GRP78 protein expression in left ventricular myocardium was analysed via immunohistochemistry (IHC) and ELISA (Fig. 3 L–N). IHC staining revealed a significant increase in GRP78-positive cells and DAB staining intensity in the HCM group (Fig. 3 L). Optical density analysis confirmed significantly higher GRP78 immunoreactivity in the HCM group compared to control, sham, AA, and HCM + AA groups (p < 0.05) (Fig. 3 M). ELISA results supported these findings, showing significantly elevated GRP78 levels in the HCM group (p < 0.05), while AA treatment reduced GRP78 expression in the HCM + AA group (Fig. 3 N). These results indicate that AA effectively alleviates ER stress induced by hypertensive cardiac remodelling in vivo. 4. In Vitro Evaluation of MAPK Signalling Pathway Components in H9C2 Cells To assess the activation of the MAPK signalling pathway in cellular hypertrophy, the total and phosphorylated forms of p38 MAPK, ERK, and JNK were examined in H9C2 cardiomyoblasts using immunofluorescence staining and ELISA (Fig. 4 ). Immunofluorescence analysis of p38 and p-p38 MAPK showed no discernible difference in the fluorescence intensity of total p38 MAPK among the experimental groups. In contrast, p-p38 MAPK fluorescence intensity was markedly elevated in cells treated with Ang II, while treatment with AA, Los, or their combination significantly diminished this increase (Fig. 4 A). Quantitative analyses by both immunofluorescence and ELISA confirmed that total p38 MAPK levels remained unchanged across all groups (p > 0.05) (Fig. 4 B–C). However, p-p38 MAPK levels were significantly increased in the Ang II group and were effectively suppressed by AA and/or Los treatment (p < 0.001) (Fig. 4 D). Consistent with these findings, the p-p38/p38 ratio was highest in the Ang II group and was significantly reduced by AA and losartan treatments (p 0.05) (Fig. 4 G–H). However, p-ERK levels were significantly elevated in Ang II-treated cells and were substantially attenuated by AA, Los, or combination therapy (p < 0.01) (Fig. 4 I). The p-ERK/ERK ratio was also significantly increased in the Ang II group and normalized following AA and/or Los treatment (p < 0.001) (Fig. 4 J). Similar trends were observed for JNK. Total JNK expression did not significantly differ between groups (Fig. 4 L–M). Nevertheless, p-JNK fluorescence intensity was significantly increased in the Ang II group (p < 0.001), and this effect was significantly mitigated by AA and/or Los p < 0.001) (Fig. 4 N). Analysis of the p-JNK/JNK ratio revealed a marked increase in the Ang II group compared to the control, Ang II + AA, Los + Ang II, and Los + Ang II + AA groups (p < 0.05) (Fig. 4 O). Collectively, these results demonstrate that Ang II activates the MAPK signalling pathway by enhancing the phosphorylation of p38, ERK, and JNK. AA significantly suppresses this activation, underscoring its potential as a modulator of MAPK-mediated hypertrophic signalling. 5. In Vivo Assessment of MAPK Signalling Pathway Activation To evaluate the activation status of the MAPK signaling cascade in vivo, immunohistochemical staining and ELISA were employed to assess the total and phosphorylated forms of p38 MAPK, ERK, and JNK in left ventricular myocardium (Fig. 5 ). Immunohistochemical analysis showed no significant differences in total p38 MAPK staining intensity among the experimental groups, a finding corroborated by ELISA quantification in total heart tissue (Fig. 5 A–C). However, p-p38 MAPK levels were significantly elevated in the HCM group compared to all other groups (p < 0.01), as determined by image analysis of staining intensity (Fig. 5 D). Furthermore, the ratio of p-p38 MAPK to total p38 MAPK was also significantly increased in the HCM group (p < 0.01), indicating activation of the p38 MAPK pathway (Fig. 5 E). Similarly, no significant differences were detected in total ERK expression across groups by either IHC or ELISA (Fig. 5 F–H). In contrast, p-ERK staining intensity was markedly higher in the HCM group compared to all other groups (p < 0.001), and this elevation was reflected in the p-ERK/ERK ratio, which was significantly increased in the HCM group (p < 0.01) (Fig. 5 I–J). These results suggest that ERK phosphorylation is a prominent feature of MAPK pathway activation in the hypertrophic myocardium. Total JNK levels showed no significant variation among groups based on both IHC and ELISA analyses (Fig. 5 K–M). However, p-JNK levels were significantly increased in the HCM group compared to all other groups (p < 0.001), as determined by staining intensity (Fig. 5 N). Correspondingly, the p-JNK/JNK ratio was significantly elevated in the HCM group (p < 0.01), further confirming activation of the JNK arm of the MAPK signalling pathway (Fig. 5 O). Collectively, these data demonstrate that while total protein levels of p38 MAPK, ERK, and JNK remain unchanged in hypertrophic myocardium, phosphorylation of these kinases is significantly upregulated. These findings confirm the activation of the MAPK signalling cascade, particularly in response to Ang II-mediated hypertrophic stimuli in vivo. Discussion Hypertensive cardiac hypertrophy represents a major pathological adaptation to increased afterload, frequently progressing to heart failure if left unchecked [ 1 ]. The hypertrophic process is characterized by cardiomyocyte enlargement, fibrosis, and maladaptive remodelling, leading to compromised cardiac function [ 2 ]. Our study demonstrates for the first time that AA, a dipeptide derivative found in edible plants and marine fungi, mitigates Ang II-induced hypertrophy and ER stress both in vitro and in a 2K1C rat model. These findings underscore the cardioprotective potential of AA via modulation of MAPK signaling and ER stress pathways, providing novel insights into its therapeutic application against hypertensive heart disease. Cardiac hypertrophy is a compensatory mechanism in response to elevated hemodynamic stress, prominently mediated by Ang II signaling [ 3 ]. Ang II promotes hypertrophic growth through AT1R-dependent activation of downstream signaling cascades, notably the MAPK pathway, resulting in pathological remodelling [ 4 ]. Consistent with previous findings, our study revealed that Ang II markedly increased H9C2 cell size, ANP levels, and total protein content, confirming successful hypertrophic induction. For instance, a study investigated the effects of Ang II on H9C2 cells and found that treatment with 1 µmol/L Ang II for 24 hours resulted in marked increases in cell surface area, total protein synthesis, and mRNA expression levels of hypertrophic markers such as ANP and brain natriuretic peptide (BNP) [ 20 ]. Treatment with AA significantly attenuated Ang II-induced hypertrophy, reducing both cellular surface area and ANP expression. Total protein content, another hallmark of hypertrophic remodelling, was also significantly reduced following AA treatment. The suppression of ANP expression is critical, as elevated ANP levels are a compensatory response to hemodynamic stress but are indicative of pathological remodelling [ 21 ]. These findings are consistent with the anti-inflammatory and vasoprotective effects of AA reported in hypertensive models [ 11 ], suggesting a direct anti-hypertrophic role. The 2K1C model recapitulates human renovascular hypertension and its associated cardiac remodelling [ 16 ]. Consistent with previous reports, rats subjected to 2K1C developed marked hypertension, cardiac hypertrophy, fibrosis, and elevated Ang II levels. Our findings revealed that AA administration ameliorated these pathological changes, significantly lowering SBP and DBP. Notably, serum Ang II concentrations were markedly reduced in AA-treated rats, suggesting an upstream modulation of the RAAS system. This reduction in Ang II levels is significant because Ang II not only promotes hypertension but also induces oxidative stress, inflammation, and fibrosis, contributing to the progression of cardiac hypertrophy [ 22 ]. Histological analysis demonstrated decreased cardiomyocyte cross-sectional area and reduced collagen deposition in AA-treated hearts, indicative of reduced hypertrophy and fibrosis. Given that fibrosis stiffens the myocardium and impairs diastolic function [ 23 ], the anti-fibrotic effect of AA suggests potential benefits in preventing heart failure progression. The reduction in heart weight and heart weight-to-body weight ratios in AA-treated animals corroborates the anti-hypertrophic efficacy observed histologically. The observed decrease in total myocardial protein content, as well as lowered ANP levels, reinforces the anti-remodelling effect of AA. ANP, a marker of myocardial stress, is upregulated in response to volume overload and hypertrophy [ 24 ]; thus, its normalization by AA treatment reflects a reversal of pathological remodelling. Activation of MAPK pathways, including p38, ERK1/2, and JNK, plays a pivotal role in pathological hypertrophic responses [ 7 , 8 ]. Our results demonstrated that Ang II stimulation increased phosphorylation of p38, ERK, and JNK in H9C2 cells without affecting total protein levels. This selective upregulation of phosphorylated MAPKs aligns with previous observations that MAPK activation, rather than expression, is key to hypertrophic signaling [ 5 ]. AA treatment significantly reduced phosphorylation levels of all three MAPK branches, restoring them close to baseline. The inhibition of MAPK phosphorylation by AA is critical because these kinases regulate transcription factors such as NF-κB and AP-1, which are responsible for the expression of pro-hypertrophic genes [ 25 ]. By modulating these signalling cascades, AA effectively prevents the transcriptional upregulation of hypertrophic genes. Importantly, the inhibition of MAPK signaling pathways in vivo corroborates findings in other models of cardiac hypertrophy where blockade of MAPK cascades attenuated hypertrophy and improved cardiac function [ 26 ]. ER stress, resulting from the accumulation of unfolded proteins, is a well-documented contributor to cardiac hypertrophy and failure [ 6 ]. In our in vitro model, TM-induced ER stress increased GRP78 expression, a hallmark of the UPR, which was further exacerbated by Ang II. Importantly, AA significantly reduced GRP78 levels, comparable to TUDCA, a known ER stress inhibitor. These findings suggest that AA not only attenuates hypertrophy via MAPK inhibition but also mitigates ER stress, potentially by enhancing protein folding capacity or facilitating proteostasis. Previous studies have shown that prolonged ER stress leads to activation of pro-apoptotic pathways, such as CHOP-mediated apoptosis, contributing to cardiomyocyte loss and cardiac dysfunction [ 27 ]. Morphological analysis confirmed that ER stress exacerbates hypertrophic changes, which were alleviated by AA treatment. The ability of AA to modulate ER stress pathways positions it as a unique therapeutic candidate targeting multiple hypertrophic mechanisms simultaneously. Moreover, the reduction in ER stress markers suggests that AA may preserve protein homeostasis, thereby protecting cardiomyocytes from stress-induced apoptosis and maladaptive remodelling. Current anti-hypertrophic therapies, such as RAAS inhibitors, beta-blockers, and mineralocorticoid receptor antagonists, primarily target neurohormonal activation but do not directly modulate intracellular stress responses like ER stress [ 28 ]. Although effective in reducing mortality, these therapies often fail to completely reverse structural cardiac changes once hypertrophy is established. AA's dual-action profile offers a potential therapeutic advantage by concurrently targeting upstream and downstream pathological processes. By mitigating both MAPK pathway activation and ER stress, AA may halt the feed-forward cycle of hypertrophy and fibrosis more effectively than current single-target therapies. This multi-targeted approach is particularly advantageous in complex diseases such as hypertensive heart disease, where multiple signalling pathways are dysregulated. The precise molecular targets of AA remain to be elucidated. Its structure, comprising N-benzoylphenylalanine and phenylalanine residues [ 10 ], suggests potential interactions with intracellular signaling molecules or chaperones involved in the UPR and MAPK cascades. Proteomic and transcriptomic studies could elucidate these interactions and identify key mediators of AA’s effects. Furthermore, additional investigations into AA's effects on oxidative stress and autophagy pathways, both of which are implicated in hypertensive cardiac remodelling, could provide a more comprehensive understanding of its cardioprotective mechanisms [ 29 ]. Future studies should also focus on dose-response relationships, long-term efficacy, and safety profiles in larger animal models. Given the chronic nature of hypertension and its associated cardiac complications, it is imperative to assess the long-term effects of AA treatment on cardiac structure and function. Additionally, studies investigating the combination of AA with existing therapies such as RAAS inhibitors could reveal synergistic effects, offering new avenues for combination therapy in hypertensive heart disease. An important aspect of our findings is the distinction between the mechanisms observed in vitro versus in vivo. In cell-based experiments, AA treatment appears to block Ang II signaling at the receptor or post-receptor level, inhibiting downstream MAPK activation and ER stress. In contrast, in the in vivo model, AA leads to a significant reduction in circulating Ang II levels, suggesting an effect on the synthesis or release of Ang II itself. At first glance, these may seem like two separate phenomena—one targeting receptor-mediated signaling, the other modulating systemic RAAS activity. However, we propose that these actions are not contradictory but rather reflect a cohesive, multi-level mechanism of AA’s cardioprotective effects. AA may exert local effects within cardiomyocytes while simultaneously influencing systemic regulators of Ang II production through vascular or renal pathways. This integrated mode of action provides a more complete understanding of how AA mitigates hypertensive cardiac remodeling, unifying both cellular and systemic effects into a single, biologically plausible therapeutic narrative. Several pharmacological agents and natural compounds exhibit dual actions similar to AA, targeting both systemic RAAS activity and intracellular hypertrophic signaling. ARBs like losartan and ACE inhibitors reduce Ang II levels in vivo while inhibiting MAPK pathways in vitro [ 30 ]. Natural compounds such as resveratrol and berberine lower blood pressure and Ang II, while also blocking Ang II-induced hypertrophy and ER stress in cardiomyocytes [ 31 – 33 ]. Given the increasing global burden of hypertension-induced cardiac hypertrophy and the limited efficacy of existing therapies in reversing structural cardiac changes [ 24 ], AA represents a promising candidate for adjunctive therapy. Its ability to modulate multiple pathological pathways simultaneously could translate into superior therapeutic outcomes for patients with hypertensive heart disease. Moreover, the natural origin of AA, found in edible plants and marine-derived fungi, may offer an advantage in terms of biocompatibility and reduced side effect profiles compared to synthetic pharmacological agents. However, comprehensive toxicological evaluations are necessary to confirm its safety profile. This study has several limitations. First, only a single dose of AA was tested in vivo; dose-response studies are warranted to establish optimal therapeutic windows. Second, long-term outcomes and potential off-target effects of AA were not assessed. Third, molecular docking and target validation studies are needed to confirm the direct interactions of AA with MAPK or ER stress components. Additionally, the lack of direct measurement of other stress pathways such as oxidative stress or autophagy limits our understanding of AA’s full spectrum of action. Future studies should also assess the effects of AA in female animal models, as sex differences in hypertensive cardiac remodeling have been reported [ 34 ]. In conclusion, 0ur study provides compelling evidence that AA mitigates hypertensive cardiac hypertrophy through inhibition of MAPK signalling and ER stress both in vitro and in vivo. These findings position AA as a promising therapeutic agent warranting further investigation for the treatment of hypertensive heart disease. Comprehensive preclinical studies are necessary to validate these findings and to facilitate the clinical translation of AA for use in patients with hypertension-induced cardiac hypertrophy. Abbreviations AA, Aurantiamide acetate ANP Atrial Natriuretic Peptide Ang II Angiotensin II AT1R Angiotensin II type 1 receptor CVF Collagen Volume Fraction DBP Diastolic Blood Pressure DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl sulfoxide eNOS Endothelial Nitric Oxide Synthase ELISA Enzyme-Linked Immunosorbent Assay ER Endoplasmic Reticulum ERK Extracellular signal-regulated kinase FBS Fetal Bovine Serum GRP78 Glucose-Regulated Protein 78 H9C2 Rat Cardiomyoblast Cell Line HCM Hypertrophic Cardiomyopathy IHC Immunohistochemistry JNK c-Jun N-terminal kinase MAPK Mitogen-Activated Protein Kinase MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NaCl Sodium Chloride OD Optical Density PBS Phosphate Buffered Saline RAAS Renin-Angiotensin-Aldosterone System SBP Systolic Blood Pressure SD Standard Deviation TM Tunicamycin TUDCA Tauroursodeoxycholic Acid UPR Unfolded Protein Response. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Ethics approval This study was reviewed and approved by Akdeniz University Experimental Animals Application and Research Center, Animal Experiments Local Ethics Committee (Ethics Committee Approval No: 6; Date: 05.02.2024; Protocol No: 1677/2024.02.003) Clinical trial number Not applicable Consent to participate Not applicable Consent for publication Not applicable Funding This work was supported by the Akdeniz University Research Foundation (Grant No: TDK-2024-6565) and the Scientific and Technological Research Council of Türkiye (TÜBİTAK) under Project No: 124S301. Author Contribution The authors declare that all data were generated in-house and that no paper mill was used. TÇ, carried out animal studies, investigation, validation, formal analysis, reviewing, and editing. MA, conceptualization, funding acquisition, supervision, writing, reviewing, and editing. EK, carried out histological studies. HTB, carried out pathological studies. ÇY, carried out cell culture, animal studies, immunohistochemical measurements and analysed data. AÖÜ, carried out cell culture, animal studies, immunohistochemical measurements and analysed data. Acknowledgement The authors would like to acknowledge Dr. Gamze Tanrıöver for her valuable advice and general guidance during the project Availability of data and material Data obtained and analysed are available from the corresponding author on reasonable request. Code availability Not applicable References Marian AJ, Braunwald E. Hypertrophic Cardiomyopathy: Genetics, Pathogenesis, Clinical Manifestations, Diagnosis, and Therapy. Circ Res. 2017 Sep 15;121(7):749-770. doi: 10.1161/CIRCRESAHA.117.311059. PMID: 28912181; PMCID: PMC5654557. Saheera S, Krishnamurthy P. Cardiovascular Changes Associated with Hypertensive Heart Disease and Aging. Cell Transplant. 2020 Jan-Dec;29:963689720920830. doi: 10.1177/0963689720920830. PMID: 32393064; PMCID: PMC7586256. Carter K, Shah E, Waite J, Rana D, Zhao ZQ. Pathophysiology of Angiotensin II-Mediated Hypertension, Cardiac Hypertrophy, and Failure: A Perspective from Macrophages. Cells. 2024 Dec 4;13(23):2001. doi: 10.3390/cells13232001. PMID: 39682749; PMCID: PMC11640308. Bhullar SK, Dhalla NS. Angiotensin II-Induced Signal Transduction Mechanisms for Cardiac Hypertrophy. Cells. 2022 Oct 22;11(21):3336. doi: 10.3390/cells11213336. PMID: 36359731; PMCID: PMC9657342. Darling NJ, Cook SJ. The role of MAPK signalling pathways in the response to endoplasmic reticulum stress. Biochim Biophys Acta. 2014 Oct;1843(10):2150-63. doi: 10.1016/j.bbamcr.2014.01.009. PMID: 24440275. An Y, Wang X, Guan X, Yuan P, Liu Y, Wei L, Wang F, Qi X. Endoplasmic reticulum stress-mediated cell death in cardiovascular disease. Cell Stress Chaperones. 2024 Feb;29(1):158-174. doi: 10.1016/j.cstres.2023.12.003. PMID: 38295944; PMCID: PMC10939083. Rose BA, Force T, Wang Y. Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale. Physiol Rev. 2010 Oct;90(4):1507-46. doi: 10.1152/physrev.00054.2009. PMID: 20959622; PMCID: PMC3808831. Yao Y, Lu Q, Hu Z, Yu Y, Chen Q, Wang QK. A non-canonical pathway regulates ER stress signaling and blocks ER stress-induced apoptosis and heart failure. Nat Commun. 2017 Jul 25;8(1):133. doi: 10.1038/s41467-017-00171-w. PMID: 28743963; PMCID: PMC5527107. Ghorani V, Saadat S, Khazdair MR, Gholamnezhad Z, El-Seedi H, Boskabady MH. Phytochemical Characteristics and Anti-Inflammatory, Immunoregulatory, and Antioxidant Effects of Portulaca oleracea L.: A Comprehensive Review. Evid Based Complement Alternat Med. 2023 Aug 31;2023:2075444. doi: 10.1155/2023/2075444. PMID: 37693918; PMCID: PMC10484659. Suhas R, Channe Gowda D. Structure-based rationale design and synthesis of aurantiamide acetate analogues - towards a new class of potent analgesic and anti-inflammatory agents. Chem Biol Drug Des. 2012 May;79(5):850-62. doi: 10.1111/j.1747-0285.2012.01331.x. PMID: 22251852. Aslan M, Basralı F, Ülker P, Barut Z, Yılmaz Ç, Çeker T, Özen N, Öztüzün A, Elpek Ö. Effects of aurantiamide on a rat model of renovascular arterial hypertension. Pflugers Arch. 2023 Oct;475(10):1177-1192. doi: 10.1007/s00424-023-02850-8. PMID: 37582694; PMCID: PMC10499692. Hernández JS, Barreto-Torres G, Kuznetsov AV, Khuchua Z, Javadov S. Crosstalk between AMPK activation and angiotensin II-induced hypertrophy in cardiomyocytes: the role of mitochondria. J Cell Mol Med. 2014 Apr;18(4):709-20. doi: 10.1111/jcmm.12220. PMID: 24444314; PMCID: PMC3981893. Thimm C, Erichsen L, Wruck W, Adjaye J. Unveiling Angiotensin II and Losartan-Induced Gene Regulatory Networks Using Human Urine-Derived Podocytes. Int J Mol Sci. 2023 Jun 23;24(13):10551. doi: 10.3390/ijms241310551. PMID: 37445727; PMCID: PMC10341883. Afşar E, Kırımlıoglu E, Çeker T, Yılmaz Ç, Demir N, Aslan M. Effect of ER stress on sphingolipid levels and apoptotic pathways in retinal pigment epithelial cells. Redox Biol. 2020 Feb;30:101430. doi: 10.1016/j.redox.2020.101430. PMID: 31978676; PMCID: PMC6976939. Yang Y, Mbikyo MB, Zhang J, Zhang Y, Zhang N, Li Z. The lncRNA MIAT regulates CPT-1a mediated cardiac hypertrophy through m6A RNA methylation reading protein Ythdf2. Cell Death Discov. 2022 Apr 5;8(1):167. doi: 10.1038/s41420-022-00977-8. PMID: 35383152; PMCID: PMC8983679. Iampanichakul M, Poasakate A, Potue P, Rattanakanokchai S, Maneesai P, Prachaney P, Settheetham-Ishida W, Pakdeechote P. Nobiletin resolves left ventricular and renal changes in 2K-1C hypertensive rats. Sci Rep. 2022 Jun 3;12(1):9289. doi: 10.1038/s41598-022-13513-6. PMID: 35662276; PMCID: PMC9166784. Cooper STE, Westaby JD, Haines ZHR, Malone GO, Sheppard MN, Meijles DN. Of Mouse and Man: Cross-Species Characterization of Hypertensive Cardiac Remodeling. Int J Mol Sci. 2022 Jul 12;23(14):7709. doi: 10.3390/ijms23147709. PMID: 35887055; PMCID: PMC9323458. Daunoravicius D, Besusparis J, Zurauskas E, Laurinaviciene A, Bironaite D, Pankuweit S, Plancoulaine B, Herlin P, Bogomolovas J, Grabauskiene V, Laurinavicius A. Quantification of myocardial fibrosis by digital image analysis and interactive stereology. Diagn Pathol. 2014 Jun 9;9:114. doi: 10.1186/1746-1596-9-114. PMID: 24912374; PMCID: PMC4072260. Gomori G. A rapid one-step trichrome stain. Am J Clin Pathol. 1950 Jul;20(7):661-4. doi: 10.1093/ajcp/20.7_ts.661. PMID: 15432364. Han J, Li J, Liu L, Li K, Zhang C, Han Y. 20-HETE mediates Ang II-induced cardiac hypertrophy via ROS and Ca2+ signaling in H9c2 cells. Sci Rep. 2025 Jan 17;15(1):2342. doi: 10.1038/s41598-025-85992-2. PMID: 39825084; PMCID: PMC11742049. Guan XH, Hong X, Zhao N, Liu XH, Xiao YF, Chen TT, Deng LB, Wang XL, Wang JB, Ji GJ, Fu M, Deng KY, Xin HB. CD38 promotes angiotensin II-induced cardiac hypertrophy. J Cell Mol Med. 2017 Aug;21(8):1492-1502. doi: 10.1111/jcmm.13076. PMID: 28296029; PMCID: PMC5542907. Mehta PK, Griendling KK. Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol. 2007 Jan;292(1):C82-97. doi: 10.1152/ajpcell.00287.2006. PMID: 16870827. Frangogiannis NG. Cardiac fibrosis. Cardiovasc Res. 2021 May 25;117(6):1450-1488. doi: 10.1093/cvr/cvaa324. PMID: 33135058; PMCID: PMC8152700. Cavallero S, González GE, Puyó AM, Rosón MI, Pérez S, Morales C, Hertig CM, Gelpi RJ, Fernández BE. Atrial natriuretic peptide behaviour and myocyte hypertrophic profile in combined pressure and volume-induced cardiac hypertrophy. J Hypertens. 2007 Sep;25(9):1940-50. doi: 10.1097/HJH.0b013e3282435b1e. PMID: 17762660. Sugden PH, Clerk A. "Stress-responsive" mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res. 1998 Aug 24;83(4):345-52. doi: 10.1161/01.res.83.4.345. PMID: 9721691. Bueno OF, Molkentin JD. Involvement of extracellular signal-regulated kinases 1/2 in cardiac hypertrophy and cell death. Circ Res. 2002 Nov 1;91(9):776-81. doi: 10.1161/01.res.0000038488.38975.1a. PMID: 12411391. Szegezdi E, Logue SE, Gorman AM, Samali A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006 Sep;7(9):880-5. doi: 10.1038/sj.embor.7400779. PMID: 16953201; PMCID: PMC1559676. Kittleson MM. A Clinician's Guide to the 2022 ACC/AHA/HFSA Guideline for the Management of Heart Failure. J Card Fail. 2022 May;28(5):831-834. doi: 10.1016/j.cardfail.2022.03.346. PMID: 35378258. Nishida K, Otsu K. Autophagy during cardiac remodeling. J Mol Cell Cardiol. 2016 Jun;95:11-8. doi: 10.1016/j.yjmcc.2015.12.003. Epub 2015 Dec 8. PMID: 26678624. Ferrario CM, Mullick AE. Renin angiotensin aldosterone inhibition in the treatment of cardiovascular disease. Pharmacol Res. 2017 Nov;125(Pt A):57-71. doi: 10.1016/j.phrs.2017.05.020. Epub 2017 May 29. PMID: 28571891; PMCID: PMC5648016. Ma E, Wu C, Chen J, Wo D, Ren DN, Yan H, Peng L, Zhu W. Resveratrol prevents Ang II-induced cardiac hypertrophy by inhibition of NF-κB signaling. Biomed Pharmacother. 2023 Sep;165:115275. doi: 10.1016/j.biopha.2023.115275. Epub 2023 Aug 2. PMID: 37541173. Kang DG, Sohn EJ, Kwon EK, Han JH, Oh H, Lee HS. Effects of berberine on angiotensin-converting enzyme and NO/cGMP system in vessels. Vascul Pharmacol. 2002 Dec;39(6):281-6. doi: 10.1016/s1537-1891(03)00005-3. PMID: 14567065. Li MH, Zhang YJ, Yu YH, Yang SH, Iqbal J, Mi QY, Li B, Wang ZM, Mao WX, Xie HG, Chen SL. Berberine improves pressure overload-induced cardiac hypertrophy and dysfunction through enhanced autophagy. Eur J Pharmacol. 2014 Apr 5;728:67-76. doi: 10.1016/j.ejphar.2014.01.061. Epub 2014 Feb 6. PMID: 24508518. Regitz-Zagrosek V, Kararigas G. Mechanistic Pathways of Sex Differences in Cardiovascular Disease. Physiol Rev. 2017 Jan;97(1):1-37. doi: 10.1152/physrev.00021.2015. PMID: 27807199. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 07 Sep, 2025 Reviews received at journal 25 Aug, 2025 Reviews received at journal 14 Aug, 2025 Reviewers agreed at journal 12 Aug, 2025 Reviews received at journal 03 Aug, 2025 Reviewers agreed at journal 31 Jul, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers invited by journal 29 Jul, 2025 Editor assigned by journal 21 Jun, 2025 Submission checks completed at journal 21 Jun, 2025 First submitted to journal 20 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6939373","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":494920699,"identity":"bf59655e-f324-4582-a423-b7b1aae98ef2","order_by":0,"name":"Tuğçe Çeker","email":"","orcid":"","institution":"Akdeniz University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tuğçe","middleName":"","lastName":"Çeker","suffix":""},{"id":494920700,"identity":"f525430f-2671-4e00-b21c-cc728eee4085","order_by":1,"name":"Mutay Aslan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYBADGQYG5gOMDWwMDGwgLg9excwMUDVsCShaJIjQwmMA1sJASIvujPyDn3lq6nj4Z/d8/DijzCaPj/8A44O3bQx15g3YtZjdSGaW5jl2mEfiztnNkhvOpRWzMRxgNpzbxiAhcwCnFgZpHrYDPAw3cjdIPmw7nNgGdJ40L1ALLpeBbPnN86+OR/5GzuOfD9v+J7YxM7D/JqAFZCYzj8GNHDbJjW0HEtvYGNiY8Wo589jMcm7fYR7DG2lmljPOJSe28TA2S845JyE5A5eW44mPb7z5VicndyP58c2eMrvE+f2HD354U2bDjydiGJjQ4pqxgQFvTIKU/MArPQpGwSgYBSMeAAAyUFWyAiEOUwAAAABJRU5ErkJggg==","orcid":"","institution":"Akdeniz University Faculty of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Mutay","middleName":"","lastName":"Aslan","suffix":""},{"id":494920701,"identity":"5832a1a6-9223-44af-aed2-385bc2fcd372","order_by":2,"name":"Esma Kırımlıoğlu","email":"","orcid":"","institution":"Akdeniz University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Esma","middleName":"","lastName":"Kırımlıoğlu","suffix":""},{"id":494920702,"identity":"6afefcf8-689f-4089-a6cc-bd4362c5ee58","order_by":3,"name":"Hazal Tuzcu Balaban","email":"","orcid":"","institution":"Akdeniz University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hazal","middleName":"Tuzcu","lastName":"Balaban","suffix":""},{"id":494920703,"identity":"e99e08b9-f055-48a4-b6a4-486cf859af76","order_by":4,"name":"Çağatay Yılmaz","email":"","orcid":"","institution":"Akdeniz University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Çağatay","middleName":"","lastName":"Yılmaz","suffix":""},{"id":494920704,"identity":"89cfe763-e753-45e5-804e-a74986918832","order_by":5,"name":"Aleyna Öztüzün Ün","email":"","orcid":"","institution":"Akdeniz University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Aleyna","middleName":"Öztüzün","lastName":"Ün","suffix":""}],"badges":[],"createdAt":"2025-06-20 13:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6939373/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6939373/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88240982,"identity":"1eeaf16a-084f-4dd8-b7c3-bae426ccf264","added_by":"auto","created_at":"2025-08-04 11:15:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1745019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of cell viability and cellular cardiac hypertrophy (A) \u003c/strong\u003eCells were treated with angiotensin II (Ang II, 0.01–10 µM) for 24 hours. Results from eight independent experiments are expressed as mean ± SD. Statistical comparisons were carried out using the Kruskal-Wallis test followed by Dunn’s post hoc analysis. No statistically significant differences were detected. \u003cstrong\u003e(B) \u003c/strong\u003eCells were treated with aurantiamide acetate (AA, 3.125–100 µM) or dimethyl sulfoxide (DMSO, 1 µl/ml) for 24 hours. Data from eight separate experiments are shown as mean ± SD. One-way ANOVA followed by Tukey’s test was used for statistical analysis. *p \u0026lt; 0.05 compared to all groups. \u003cstrong\u003e(C) \u003c/strong\u003eCells were treated with losartan (Los, 5 and 10 µM) or 1 µl/ml DMSO for 25 hours. Results, obtained from eight independent experiments, are presented as mean ± SD. Statistical significance was evaluated using Kruskal-Wallis test with Dunn’s multiple comparison test. No significant group differences were noted. \u003cstrong\u003e(D) \u003c/strong\u003eCo-evaluation of 1 µl/ml, DMSO, 1 µM Ang II and 6.25 µM AA, following 24-hour incubation in H9C2 cells. 10 µM losartan was applied to cells 1 hour prior to the incubation period. Data represents eight replicates, with values expressed as mean ± SD. Kruskal-Wallis test and Dunn’s post hoc test revealed no significant differences.\u003cstrong\u003e (E) \u003c/strong\u003eRepresentative images of cell area by phalloidin staining after 24 hours total incubation. Applied doses were 1 µl/ml DMSO, 1 µM Ang II and 6.25 µM AA. 10 µM losartan was applied to cells 1 hour prior to the incubation period. Scale bar: 200 µm. \u003cstrong\u003e(F) \u003c/strong\u003eData represents eight replicates, with values expressed as mean ± SD. Kruskal-Wallis test and Dunn’s post hoc test revealed no significant differences. \u003cstrong\u003e(G) \u003c/strong\u003eMeasurement of\u003cstrong\u003e \u003c/strong\u003eANP protein levels in H9C2 cells. Results from five replicates are expressed as mean ± SD. Statistical evaluation was performed using one-way ANOVA with Tukey’s post hoc test. *p \u0026lt; 0.05 vs. control, DMSO, Los, Los + Ang II, and Los + Ang II + AA groups. \u003cstrong\u003e(H) \u003c/strong\u003eTotal protein levels in cell lysates Data are based on five replicates and shown as mean ± SD. One-way ANOVA followed by Tukey’s test was used for statistical comparison. *p \u0026lt; 0.001 compared to all groups.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6939373/v1/d1667eaff97f333c686384cb.jpg"},{"id":88240981,"identity":"120861aa-d4b3-47f0-9fba-5e9a8eeae798","added_by":"auto","created_at":"2025-08-04 11:15:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":563750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of ER stress on cellular cardiac hyperthrophy.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Assessment of cell viability in cells treated with tunicamycin (TM, 0.5–10 µg/ml) or dimethyl sulfoxide (DMSO, 1 µl/ml) for 24 hours. Results are based on eight independent replicates and are presented as mean ± SD. Statistical comparisons were made using one-way ANOVA followed by Tukey’s post hoc test. *p \u0026lt; 0.05 versus control, DMSO, and 0.5 µg/ml TM groups. \u003cstrong\u003e(B)\u003c/strong\u003e Cells were treated with increasing concentrations of tauroursodeoxycholic acid (TUDCA; 0.06–4 mM) for 12 hours. Data from eight independent experiments are shown as mean ± SD. One-way ANOVA followed by Tukey’s test was used to assess significance. *p \u0026lt; 0.001 compared to all other groups. \u003cstrong\u003e(C)\u003c/strong\u003e Co-evaluation of 1 µl/ml DMSO, 0.5 µg/ml TM, 1 µM Ang II, and 6.25 µM AA doses in H9C2 cells. 1 mM TUDCA was applied to cells 12 hours after the start of the incubation period and the total incubation period was 24 hours. Results from 6–8 experiments are expressed as mean ± SD. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s post hoc comparison. *p \u0026lt; 0.05 versus control, DMSO, and TM + AA groups.\u003cstrong\u003e (D)\u003c/strong\u003e Representative immunofluorescence images of GRP78 staining in H9C2 cells following treatment with DMSO (1 µl/ml), TM (0.5 µg/ml), AA (6.25 µM), Ang II (1 µM), and TUDCA (1 mM). TUDCA was administered 12 hours into the incubation period, which lasted for 24 hours in total. Scale bar: 200 µm. \u003cstrong\u003e(E)\u003c/strong\u003e Quantification of GRP78 fluorescence staining by Image J (version 1.54m). Ten measurements per group were used to calculate mean ± SD. One-way ANOVA followed by Tukey’s test was applied. *p \u0026lt; 0.05 versus all groups; #p \u0026lt; 0.05 compared to control, DMSO, TM + TUDCA, TM + AA, and TM + Ang II + AA groups. \u003cstrong\u003e(F)\u003c/strong\u003e Measurement of GRP78 protein levels in H9C2 cells. Data represent three replicates and are given as mean ± SD. One-way ANOVA followed by Tukey’s multiple comparison test was used. *p \u0026lt; 0.05 compared to all other groups; #p \u0026lt; 0.05 versus control, DMSO, TM + TUDCA, TM + AA, TM + Ang II + TUDCA, and TM + Ang II + AA groups. \u003cstrong\u003e(G)\u003c/strong\u003ePhalloidin-stained images illustrate cell morphology following 24-hour treatment with DMSO (1 µl/ml), TM (0.5 µg/ml), Ang II (1 µM), AA (6.25 µM), and TUDCA (1 mM). TUDCA was administered 12 hours after treatment began. Scale bar: 200 µm.\u003cstrong\u003e (H)\u003c/strong\u003e Cell surface area quantification was performed using ImageJ (version 1.54m), with 17–20 measurements per group. Results are presented as mean ± SD. Statistical comparisons were made using Kruskal-Wallis test and Dunn’s post hoc test. *p \u0026lt; 0.01 vs. control, DMSO, and TM + Ang II.\u003cstrong\u003e (I)\u003c/strong\u003eTotal protein levels in cell lysates. Data from five replicates are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6939373/v1/eb9cd522985073dca373d13a.jpg"},{"id":88240986,"identity":"52d6b228-062a-4e46-82f5-d8d93859280c","added_by":"auto","created_at":"2025-08-04 11:15:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3879303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of hypertrophic cardiomyopathy and ER stress in vivo.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Systolic blood pressure (SBP) in rats at 4 weeks. Data are expressed as mean ± SEM (n=8). Statistical evaluation was conducted using two-way ANOVA with Tukey’s post hoc test. *, p\u0026lt;0.01, vs. week 0 within the same group. #, p\u0026lt;0.05, vs. control, Sham and AA groups within the same week. §, p\u0026lt;0.001, vs. HCM+AA group within the same week. ¶, p\u0026lt;0.05, vs. control group within the same week. \u003cstrong\u003e(B)\u003c/strong\u003e Diastolic blood pressure in rats at 4 weeks. Values are mean± SEM (n=8). Values are presented as mean ± SEM (n=8). Two-way ANOVA with Tukey’s test was used to assess statistical significance. *, p\u0026lt;0.05, vs. week 0 within the same group. #, p\u0026lt;0.01, vs. control, Sham and AA groups within the same week. §, p\u0026lt;0.001, vs. HCM+AA group within the same week. ¶, p\u0026lt;0.05, vs. control group within the same week. \u003cstrong\u003e(C)\u003c/strong\u003e Ang II levels in rat serum by ELISA measurements. Data are shown as mean ± SD (n=8). Statistical comparison was performed using one-way ANOVA with Tukey’s test. *p \u0026lt; 0.001 vs. all experimental groups.\u003cstrong\u003e (D)\u003c/strong\u003e Representative H\u0026amp;E-stained longitudinal heart sections from the left ventricle across all groups. Scale bars: 50 µm (40× magnification). \u003cstrong\u003e(E)\u003c/strong\u003e Cross-sectional area measurement in left ventricular cardiomyocytes by Fiji (ImageJ version 1.54p). Mean ± SD values are based on 10 measurements per group from 4 animals. One-way ANOVA and Tukey’s post hoc test were used. *p \u0026lt; 0.001 vs. all groups.\u003cstrong\u003e (F)\u003c/strong\u003e ANP levels in total heart tissues by ELISA measurements. Values are presented as mean ± SD (n = 7–8). Statistical significance was assessed using one-way ANOVA and Tukey’s test. *p \u0026lt; 0.05 vs. all groups.\u003cstrong\u003e (G)\u003c/strong\u003e Total protein content in heart tissues was measured using the Bradford assay. Data are mean ± SD (n = 8). One-way ANOVA with Tukey’s test was used for statistical comparisons. *p \u0026lt; 0.05 vs. all groups. \u003cstrong\u003e(H) \u003c/strong\u003eRepresentative images of left ventricular myocardium by Masson’s trichrome staining in different experimental groups. Scale bars: 50 µm (40×). \u003cstrong\u003e(I)\u003c/strong\u003e Collagen volume fraction measurements in left ventricular cardiomyocytes by Fiji (ImageJ version 1.54p). Data are expressed as mean ± SD from 10 measurements per group (n=4). Statistical analysis was carried out using one-way ANOVA and Tukey’s post hoc test. *p \u0026lt; 0.001 vs. all groups.\u003cstrong\u003e (J)\u003c/strong\u003e Heart weights of animals from different groups. Values represent mean ± SD (n = 4). Statistical significance was assessed via one-way ANOVA followed by Tukey’s test. *p \u0026lt; 0.05 vs. all groups. \u003cstrong\u003e(K)\u003c/strong\u003e Heart-to-body weight ratios for each group. Results are shown as mean ± SD (n = 4). Statistical analysis was conducted using one-way ANOVA and Tukey’s test. *p \u0026lt; 0.05 vs. control, Sham, and AA groups. \u003cstrong\u003e(L)\u003c/strong\u003eRepresentative images of GRP78 immunohistochemical staining in left ventricle myocardium. Scale bars: 50 µm (40×). \u003cstrong\u003e(M)\u003c/strong\u003e GRP78 staining intensity by Fiji (ImageJ version 1.54p). Mean ± SD values are based on 8 measurements from 4 rats per group. One-way ANOVA followed by Tukey’s post hoc test was used. *p \u0026lt; 0.05 vs. all groups. \u003cstrong\u003e(N)\u003c/strong\u003e GRP78 levels in total heart tissues by ELISA measurements. Values are reported as mean ± SD (n = 7–8). One-way ANOVA with Tukey’s test was performed. *p \u0026lt; 0.05 vs. control, Sham, and AA groups.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6939373/v1/bcaa77032e5de1f8a9fae0f9.jpg"},{"id":88240983,"identity":"7ea0a456-cd1a-44a2-9598-f4e553cfb960","added_by":"auto","created_at":"2025-08-04 11:15:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3462599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of MAPK signaling pathway components in vitro.\u003c/strong\u003e Cells were treated with DMSO (1 µl/ml), Ang II (1 µM) and AA (6.25 µM) for 24 hours. Losartan (10 µM) was applied to cells 1 hour prior to the incubation period. Scale bar: 200 µm. \u003cstrong\u003e(A)\u003c/strong\u003e Representative immunofluorescent staining of p38 MAPK and p-p38 MAPK in H9C2 cells. \u003cstrong\u003e(B)\u003c/strong\u003eQuantification of p38 MAPK signal intensity was conducted using ImageJ software (version 1.54m), based on 10 individual measurements. Results are shown as mean ± SD. One-way ANOVA followed by Tukey’s test was used for statistical evaluation. No significant differences were observed among the groups. \u003cstrong\u003e(C)\u003c/strong\u003eMeasurement of p38 MAPK protein levels in H9C2 cells. Data from 5 independent experiments are reported as mean ± SD. Statistical analysis was performed using the Kruskal-Wallis test with Dunn’s post hoc comparisons. No statistically meaningful differences were found. \u003cstrong\u003e(D) \u003c/strong\u003eQuantification of p-p38 MAPK fluorescence staining by Image J (version 1.54m). Data represents 10 individual measurements and values are given as mean ± SD. Data represent 10 measurements per group, shown as mean ± SD. One-way ANOVA followed by Tukey’s test revealed significant differences. *p \u0026lt; 0.001 compared to all groups. \u003cstrong\u003e(E) \u003c/strong\u003eRatio of p-p38 MAPK/p38 MAPK fluorescence levels in H9C2 cells. Values are based on 10 replicates and are presented as mean ± SD. One-way ANOVA followed by Tukey’s post hoc analysis was applied. *p \u0026lt; 0.001 vs. all groups. \u003cstrong\u003e(F)\u003c/strong\u003eRepresentative immunofluorescent staining of ERK and p-ERK in H9C2 cells. \u003cstrong\u003e(G)\u003c/strong\u003eQuantification of ERK fluorescence signal using ImageJ (version 1.54m), based on 8 measurements per group. Data are shown as mean ± SD. Statistical testing was done with the Kruskal-Wallis test and Dunn’s post hoc test. No significant differences were identified. \u003cstrong\u003e(H)\u003c/strong\u003e Measurement of ERK protein levels in H9C2 cells. Data from 5 independent samples are given as mean ± SD. One-way ANOVA and Tukey’s test showed no statistically significant variation across groups. \u003cstrong\u003e(I)\u003c/strong\u003e p-ERK fluorescence levels were quantified using ImageJ (version 1.54m) from 10 individual measurements. Mean ± SD values are presented. One-way ANOVA and Tukey’s post hoc test indicated a significant difference. *p \u0026lt; 0.01 compared to all groups. \u003cstrong\u003e(J)\u003c/strong\u003e Ratio of p-ERK/ERK fluorescence levels in H9C2 cells. Values are from 8 measurements per group and are expressed as mean ± SD. Statistical analysis was conducted using one-way ANOVA with Tukey’s test. *p \u0026lt; 0.001 vs. all groups; #p \u0026lt; 0.05 vs. control and DMSO.\u003cstrong\u003e (K)\u003c/strong\u003e Representative immunofluorescent staining of JNK and p-JNK in H9C2 cells. \u003cstrong\u003e(L)\u003c/strong\u003e JNK fluorescence intensity was quantified using ImageJ (version 1.54m), based on 8 measurements per group. Mean ± SD values were calculated. One-way ANOVA with Tukey’s multiple comparisons revealed no significant differences. \u003cstrong\u003e(M)\u003c/strong\u003e Measurement of JNK protein levels in H9C2 cells. Data represent 5 individual experiments and are presented as mean ± SD. Statistical evaluation was performed via Kruskal-Wallis and Dunn’s multiple comparisons. No statistically significant variation was observed. \u003cstrong\u003e(N)\u003c/strong\u003eQuantification of p-JNK fluorescence staining by Image J (version 1.54m). Data represent 8 measurements and are shown as mean ± SD. One-way ANOVA with Tukey’s test was used. *p \u0026lt; 0.001 compared to all groups.\u003cstrong\u003e (O)\u003c/strong\u003e The ratio of p-JNK/JNK fluorescence levels in H9C2 cells. Data include 8 measurements per group and are expressed as mean ± SD. Kruskal-Wallis test with Dunn’s multiple comparisons showed significance. *p \u0026lt; 0.05 vs. control, Ang II + AA, Los + Ang II, and Los + Ang II + AA groups.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6939373/v1/5d29c1ea3a38369e9d0b1b8a.jpg"},{"id":88240989,"identity":"9252351f-fe8d-498f-b88e-c6be3139a007","added_by":"auto","created_at":"2025-08-04 11:15:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3591028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of MAPK signaling pathway in vivo.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eRepresentative images of p38- and phosphorylated-p38- MAPK immunohistochemical staining in left ventricle myocardium. Scale bar: 50 µm (40× magnification). \u003cstrong\u003e(B)\u003c/strong\u003ep38 MAPK staining intensity measured by Fiji software (ImageJ version 1.54p). Results are presented as mean ± SD (n = 4), based on 14–15 measurements per group. Statistical comparisons were made using the Kruskal-Wallis test followed by Dunn’s multiple comparisons. No statistically significant group differences were found.\u003cstrong\u003e (C)\u003c/strong\u003e p38 MAPK protein levels in total heart tissues measured by ELISA. Data are shown as mean ± SD (n = 7–8). Kruskal-Wallis test with Dunn’s post hoc analysis indicated no significant differences between groups. \u003cstrong\u003e(D)\u003c/strong\u003ep-p38 MAPK staining intensity determined by Fiji software. Results are presented as mean ± SD (n = 4), derived from 13–15 regions per group. Statistical analysis was carried out with Kruskal-Wallis followed by Dunn’s test. *p \u0026lt; 0.01 vs. all groups. \u003cstrong\u003e(E)\u003c/strong\u003e Ratio of p-p38 MAPK/p38 MAPK staining. Values are shown as mean ± SD (n = 4), based on 13–14 individual measurements per group. Kruskal-Wallis test and Dunn’s post hoc comparisons revealed significance. *p \u0026lt; 0.01 vs. all groups. \u003cstrong\u003e(F)\u003c/strong\u003e Representative images of ERK and phosphorylated-ERK immunohistochemical staining in left ventricle myocardium. Scale bars: 50 µm (40×). \u003cstrong\u003e(G)\u003c/strong\u003e ERK staining intensity measured by Fiji software. Values are mean ± SD (n=4). Data are reported as mean ± SD (n = 4), with 14–15 measurements per group. Kruskal-Wallis test and Dunn’s comparison showed no significant differences between groups. \u003cstrong\u003e(H)\u003c/strong\u003e ERK protein levels measured in total heart tissues by ELISA. Data are mean ± SD (n = 7–8). Kruskal-Wallis and Dunn’s test indicated no statistically significant variation across groups. \u003cstrong\u003e(I)\u003c/strong\u003ep-ERK staining intensity measured by Fiji software. Data represent mean ± SD (n = 4) from 14–15 measurements per group. Statistical evaluation was performed using one-way ANOVA and Tukey’s test. *p \u0026lt; 0.001 compared to all groups.\u003cstrong\u003e(J)\u003c/strong\u003e Ratio of p-ERK/ERK staining. Values are mean ± SD (n=4). Data represents 14 measurements from each group. Kruskal-Wallis and Dunn’s multiple comparisons showed significant differences. *p \u0026lt; 0.01 vs. all groups. \u003cstrong\u003e(K)\u003c/strong\u003eRepresentative images of JNK and phosphorylated-JNK immunohistochemical staining in left ventricle myocardium. Scale bars: 50 µm (40×). \u003cstrong\u003e(L) \u003c/strong\u003eJNK staining intensity measured by Fiji software. Values are shown as mean ± SD (n = 4), calculated from 14–15 measurements per group. Kruskal-Wallis test followed by Dunn’s comparison found no statistically significant changes.\u003cstrong\u003e (M) \u003c/strong\u003eJNK protein levels in total heart tissues measured by ELISA. Data are given as mean ± SD (n = 7–8). Statistical analysis using Kruskal-Wallis and Dunn’s post hoc test showed no significant differences. \u003cstrong\u003e(N) \u003c/strong\u003ep-JNK staining intensity measured by Fiji software. Data are expressed as mean ± SD (n = 4), based on 14–15 data points per group. One-way ANOVA followed by Tukey’s test revealed a significant increase. *p \u0026lt; 0.001 vs. all groups.\u003cstrong\u003e (O)\u003c/strong\u003e Ratio of p-JNK/JNK staining. Values are mean ± SD (n=4). Values are mean ± SD (n = 4), from 14 individual measurements per group. Statistical analysis using Kruskal-Wallis and Dunn’s test showed significant differences. *p \u0026lt; 0.01 vs. all groups.\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6939373/v1/f8c18e555d6985ba7addde56.jpg"},{"id":88243016,"identity":"f3cbddb8-63c0-4b2b-95b8-4db8504ab17c","added_by":"auto","created_at":"2025-08-04 11:39:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14591782,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6939373/v1/845e47ca-6180-432a-a337-f1884fe72175.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Aurantiamide Acetate Mitigates Hypertensive Cardiac Hypertrophy via Modulation of MAPK Signaling and ER Stress","fulltext":[{"header":"Key Points","content":"\u003cp\u003e\u0026bull; AA reduced cardiomyocyte hypertrophy and ER stress in H9C2 cells.\u003c/p\u003e\u003cp\u003e\u0026bull; AA suppressed MAPK pathway activation in vitro and in vivo.\u003c/p\u003e\u003cp\u003e\u0026bull; AA decreased myocardial fibrosis and improved cardiac remodelling.\u003c/p\u003e\u003cp\u003e\u0026bull; AA lowered blood pressure and serum Ang II levels in hypertensive rats.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eHypertrophic cardiomyopathy is a primary myocardial disorder characterized by excessive thickening of the myocardium, most notably the left ventricle, in the absence of abnormal loading conditions such as hypertension or valvular disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, hypertensive-induced cardiac hypertrophy shares pathological similarities with HCM, including myocardial fibrosis, cardiomyocyte hypertrophy, and adverse ventricular remodelling, ultimately predisposing individuals to heart failure, arrhythmias, and sudden cardiac death [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Understanding the molecular mechanisms underlying hypertrophy in hypertension is critical for developing targeted therapies to prevent or reverse disease progression.\u003c/p\u003e\u003cp\u003eAngiotensin II plays a pivotal role in hypertensive cardiac remodelling by promoting cardiomyocyte growth, fibrosis, and inflammation through activation of the renin-angiotensin-aldosterone system (RAAS) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Binding of Ang II to the angiotensin type 1 receptor (AT1R) initiates multiple intracellular signaling cascades, among which the MAPK pathway, including p38 MAPK, ERK, and JNK, is heavily implicated in mediating hypertrophic responses [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition to promoting hypertrophic growth, MAPK signalling has been linked to the induction of ER stress, a condition resulting from the accumulation of misfolded proteins that triggers the unfolded protein response (UPR) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Persistent ER stress exacerbates cardiac dysfunction by impairing cellular homeostasis and promoting apoptosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent studies have illuminated the intersection between MAPK activation and ER stress in driving cardiac hypertrophy, suggesting that agents targeting these pathways could offer therapeutic benefits [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Despite this knowledge, clinically effective interventions that modulate these pathways remain limited, underscoring the need for novel therapeutic strategies.\u003c/p\u003e\u003cp\u003eAurantiamide acetate, a dipeptide derivative commonly found in edible plants such as \u003cem\u003ePortulaca oleracea L.\u003c/em\u003e (purslane) and marine-derived fungi (\u003cem\u003eAspergillus\u003c/em\u003e species), has attracted attention for its diverse pharmacological properties, including anti-inflammatory, antioxidant, and anti-cancer activities [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Structurally, AA consists of N-benzoylphenylalanine and phenylalanine residues, conferring it the ability to modulate key biological pathways [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previous investigations have demonstrated that AA exerts vasoprotective effects by enhancing endothelial nitric oxide synthase (eNOS) expression, improving vascular reactivity, and reducing oxidative stress in experimental models of hypertension [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In particular, AA was shown to attenuate mean blood pressure, improve endothelial-dependent relaxation, and enhance red blood cell deformability in rats subjected to 2K1C renovascular hypertension, suggesting a multifaceted role in cardiovascular protection [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite the growing body of research highlighting the pathological roles of MAPK signaling and ER stress in cardiac hypertrophy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and the known beneficial effects of AA on vascular function, the potential cardioprotective role of AA in hypertensive HCM had not been previously explored [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Importantly, while AA's vascular effects were studied in hypertensive models, its direct impact on cardiomyocytes, particularly in the context of hypertrophic growth and ER stress modulation, remained unclear. Furthermore, there was a lack of comprehensive investigations integrating both in vitro and in vivo approaches to elucidate the molecular mechanisms underlying AA\u0026rsquo;s potential cardioprotective actions.\u003c/p\u003e\u003cp\u003eTo address these gaps, the present study was designed to evaluate the effects of AA on hypertensive cardiac hypertrophy, focusing on its ability to modulate MAPK signaling and ER stress. The study employed a two-pronged approach: first, using H9C2 cardiomyoblasts to examine the direct effects of AA on cellular hypertrophy and ER stress markers in vitro; and second, employing a 2K1C rat model to validate the in vivo cardioprotective effects of AA against hypertension-induced hypertrophic remodelling. This integrated strategy aimed to provide novel insights into the therapeutic potential of AA as a modulator of hypertrophic signaling pathways and ER stress, offering a promising avenue for the treatment of hypertensive heart disease. Therefore, we hypothesized that AA could attenuate cardiac hypertrophy by inhibiting MAPK signaling and alleviating ER stress. The primary objective of this study was to evaluate the effects of AA on hypertrophic remodelling and ER stress responses in Ang II-stimulated H9C2 cardiomyoblasts. A secondary objective was to validate these findings in vivo using a 2K1C rat model of hypertensive HCM by assessing myocardial structure, fibrosis, hypertrophy markers, and ER stress-related protein expression. Through this integrated approach, we aimed to provide new mechanistic insights into the cardioprotective potential of AA and its relevance as a therapeutic candidate for hypertensive cardiac disease.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell Culture\u003c/h2\u003e\u003cp\u003eThe rat cardiomyoblast cell line, H9C2 (ATCC\u0026reg; CRL-1446\u0026trade;) was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained in low-glucose Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM; D6046, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 1000 mg/L L-glutamine and sodium bicarbonate. The medium was further enriched with 10% (v/v) fetal bovine serum (FBS; Capricorn Scientific, FBS-11A, Ebsdorfergrund, Germany), 1% (v/v) penicillin (100 U/mL) and streptomycin (100 \u0026micro;g/mL) (Gibco, Life Technologies Corporation, 15140-122, Grand Island, NY, USA), and Amphotericin-B at a concentration of 200 \u0026micro;L/L (Gibco, Life Technologies Limited, 15290-026, Paisley, UK). The complete medium was sterilized using a 0.22 \u0026micro;m bottle-top filter and stored at 4\u0026deg;C until use. Cells were cultured at 37\u0026deg;C in a humidified incubator with 5% CO₂ and 95% air. Upon reaching approximately 80% confluence, cells were subcultured using 0.05% trypsin-EDTA (1X) (Gibco, Life Technologies Corporation, 25300054, Grand Island, NY, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInduction of Cellular Hypertrophy\u003c/h3\u003e\n\u003cp\u003eCellular hypertrophy was induced using Ang II (Sigma-Aldrich, A9525, Darmstadt, Germany) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The powdered compound was dissolved in sterile distilled water to prepare a 24 mM stock solution. Working solutions at final concentrations of 0.01, 0.1, 0.2, 1, and 10 \u0026micro;M were obtained by diluting the stock solution with cell culture medium and applied to the cells for 24 hours. To verify that the hypertrophic effects of Ang II were mediated via the angiotensin II type 1 receptor (AT1R), the selective AT1R antagonist losartan (Los; Sigma-Aldrich, SML3317, Darmstadt, Germany) was used as a negative control [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Losartan was dissolved in dimethyl sulfoxide (DMSO; Fisher Scientific, BP231-1, Leicestershire, UK) to prepare a 10 mM stock solution. Final concentrations of 5 \u0026micro;M and 10 \u0026micro;M were administered to the cells 1 hour prior to Ang II treatment.\u003c/p\u003e\n\u003ch3\u003eAurantiamide Acetate Treatment\u003c/h3\u003e\n\u003cp\u003eAurantiamide acetate (MedChemExpress, HY-N2905, Monmouth Junction, NJ, USA) was obtained in powder form and dissolved in DMSO to prepare a 100 mM stock solution. The stock was subsequently diluted with culture medium to achieve final concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 \u0026micro;M. These concentrations were applied to the cells 24 hours [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eInduction and Inhibition of Endoplasmic Reticulum Stress\u003c/h3\u003e\n\u003cp\u003eEndoplasmic reticulum stress was induced using TM (Abcam, ab120296, Cambridge, UK) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. TM was dissolved in DMSO to prepare a 10 mg/mL stock solution. This stock was diluted in cell culture medium to final concentrations of 0.5, 1, 2, 5, and 10 \u0026micro;g/mL and applied to the cells for 24 hours. Tauroursodeoxycholic acid (TUDCA; Millipore, 580549, Darmstadt, Germany), a known ER stress inhibitor, was used to attenuate TM-induced stress [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. TUDCA was dissolved in sterile 0.9% isotonic sodium chloride (NaCl) to prepare a 10 mM stock solution. Final concentrations of 0.06, 0.125, 0.5, 1, 2, and 4 mM were administered to the cells 12 hours after the initiation of TM treatment.\u003c/p\u003e\n\u003ch3\u003eCell Viability Assay\u003c/h3\u003e\n\u003cp\u003eCell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; GoldBio, T-030-1, St. Louis, MO, USA) colorimetric assay. MTT powder was dissolved in 1X phosphate-buffered saline (PBS; Sigma-Aldrich, P4417, St. Louis, MO, USA) at a concentration of 5 mg/mL, following the manufacturer\u0026rsquo;s protocol, and sterilized by filtration through a 0.22 \u0026micro;m membrane filter. H9C2 cells were seeded at a density of 5,000 cells per well in 96-well plates and incubated for 24 hours to allow adherence. Subsequently, cells were treated with Ang II, Los, AA, TM, or TUDCA at the designated concentrations and exposure times, in a final volume of 200 \u0026micro;L per well. At the end of the treatment period, MTT solution was added to each well to achieve a final concentration of 0.5 mg/mL. Plates were then incubated for 2 hours at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂, protected from light. Following incubation, the culture medium was aspirated, and the resulting formazan crystals were dissolved in 100 \u0026micro;L of DMSO. Absorbance was recorded using a spectrophotometer at 570 nm, with background subtraction at 690 nm. Cell viability was expressed as a percentage relative to the untreated control group, calculated by the formula: Cell viability (%) = (Abs_sample / Abs_control) \u0026times; 100.\u003c/p\u003e\u003cp\u003eBased on MTT cell viability assay results, non-cytotoxic and experimentally appropriate treatment groups were established as follows: Control cells were cultured in low-glucose DMEM alone; the DMSO group received 1 \u0026micro;L/mL DMSO (vehicle control) for 24 hours; hypertrophy was induced by treating cells with 1 \u0026micro;M Ang II for 24 hours (Ang II group); cells treated with 6.25 \u0026micro;M AA for 24 hours constituted the AA group, while the Ang II\u0026thinsp;+\u0026thinsp;AA group received combined treatment for 24 hours. The Los group was treated with 10 \u0026micro;M for 25 hours; in the Los\u0026thinsp;+\u0026thinsp;Ang II group, Los was administered 1 hour prior to 24-hour Ang II exposure, and in the Los\u0026thinsp;+\u0026thinsp;Ang II\u0026thinsp;+\u0026thinsp;AA group, Los pretreatment was followed by co-administration of Ang II and AA for 24 hours. ER stress was induced by treating cells with 0.5 \u0026micro;g/mL TM for 24 hours (TM group). Co-treatment groups included TM\u0026thinsp;+\u0026thinsp;Ang II, TM\u0026thinsp;+\u0026thinsp;AA, and TM\u0026thinsp;+\u0026thinsp;Ang II\u0026thinsp;+\u0026thinsp;AA, all for 24 hours. Additionally, the TM\u0026thinsp;+\u0026thinsp;TUDCA group involved a total 24-hour incubation where 1 mM TUDCA was added 12 hours post-TM treatment, while in the TM\u0026thinsp;+\u0026thinsp;Ang II\u0026thinsp;+\u0026thinsp;TUDCA group, TUDCA was administered 12 hours after combined TM and Ang II treatment.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell Surface Area Measurement\u003c/h2\u003e\u003cp\u003eCell surface area was quantified using a fluorescent phalloidin conjugate specific for F-actin filaments (Phalloidin-iFluor\u0026trade; 555 Conjugate; Santa Cruz Biotechnology, sc-363794, Dallas, TX, USA) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The lyophilized reagent was reconstituted in DMSO to prepare a 1000X stock solution, which was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use. H9C2 cells were seeded at a density of 50,000 cells per well into 8-well chamber slides (Merck Millipore, PEZGS0816, Cork, Ireland) and incubated overnight to ensure adherence. The following day, after confirming 70\u0026ndash;80% confluency and normal morphology, cells were treated with experimental media according to group-specific concentrations and incubation times. Following treatment, cells were washed twice with cold PBS and fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich, 158127, St. Louis, MO, USA) for 10 minutes. After three PBS washes, cell membranes were permeabilized using 0.2% Triton X-100 (Sigma-Aldrich, T8787) for 10 minutes, followed by five additional PBS washes. A 1X staining solution was prepared by diluting the phalloidin stock 1:1000 in PBS containing 1% BSA (Sigma-Aldrich, A8806) and incubated with cells at room temperature for 45 minutes. Nuclei were counterstained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, H-1200) and mounted with coverslips, ensuring the absence of air bubbles. Fluorescence images were acquired using an Olympus IX81 inverted microscope (Olympus Corporation, Tokyo, Japan) equipped with TRITC and DAPI filter sets at 20\u0026times; magnification. Image analysis was performed using ImageJ software (version 1.54m; NIH, Bethesda, MD, USA). Images were converted to RGB stack format, and thresholding was applied to isolate cell regions. Using the ROI Manager tool, cell boundaries were manually delineated based on F-actin signals, and the surface area (\u0026micro;m\u0026sup2;) of at least 20 individual cells per group was measured. Data was subjected to statistical analysis to evaluate differences between experimental groups.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunofluorescence Staining\u003c/h3\u003e\n\u003cp\u003eTo assess the expression of target proteins in H9C2 cells, immunofluorescence staining was performed. Cells at approximately 80% confluency were detached using 0.05% trypsin-EDTA and seeded onto 8-well chamber slides at a density of 20,000 cells per well. After overnight incubation to allow cell adhesion, confluency (70\u0026ndash;80%) and normal morphology were confirmed microscopically. Cells were then treated with the designated media corresponding to each experimental group. At the end of the treatment period, culture media were removed, and cells were washed twice with PBS. Fixation was performed with 4% paraformaldehyde for 10 minutes at room temperature, followed by three PBS washes. Cells were permeabilized using 0.2% Triton X-100 in PBS for 10 minutes and then washed five times with PBS. To block nonspecific antibody binding, cells were incubated with 5% normal goat serum (NGS; Vector Laboratories Inc., S-1000-20; Newark, CA, USA) in PBS for 30 minutes at room temperature. Subsequently, cells were incubated overnight at 4\u0026deg;C in the dark with 200 \u0026micro;L per well of the following primary antibodies, each diluted 1:200 in PBS containing 1% BSA: rabbit anti-GRP78 (Abcam, ab21685; Cambridge, MA, USA), anti-p38 MAPK (Affinity Biosciences, AF6456; Cincinnati, OH, USA), anti-phospho-p38 MAPK (Thr180/Tyr182; Affinity Biosciences, AF4001), anti-ERK1/2 (Affinity Biosciences, AF0155), anti-phospho-ERK1/2 (Thr202/Tyr204; Affinity Biosciences, AF1015), anti-JNK1/2/3 (Affinity Biosciences, AF6319), and anti-phospho-JNK1/2/3 (Thr183\u0026thinsp;+\u0026thinsp;Tyr185; Affinity Biosciences, AF3318). On the following day, cells were washed five times with PBS and incubated for 45 minutes at room temperature in the dark with a goat anti-rabbit IgG secondary antibody conjugated to Alexa Fluor\u0026reg; 488 (1:1000; Abcam, ab150077; Cambridge, UK) and F-actin counterstain using Phalloidin-iFluor\u0026trade; 555 (1X; Abcam, ab176756; Cambridge, UK), both diluted in PBS with 1% BSA at a volume of 200 \u0026micro;L per well. After three additional PBS washes, the chamber structure was removed, and nuclei were counterstained with DAPI (Vector Laboratories, H-1200; Newark, CA, USA) by adding a drop to each well and mounting coverslips without air bubbles. Fluorescence images were acquired using an Olympus IX81 inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with FITC (Alexa 488), TRITC (Phalloidin 555), and DAPI filter sets, at 20\u0026times; magnification. Image analysis was conducted using ImageJ software (version 1.54m; NIH, Bethesda, MD, USA). Fluorescently labeled cell regions were manually delineated for each group, and fluorescence intensity was quantified using the Integrated Density function. Corrected Total Cell Fluorescence (CTCF) was calculated using the formula: CTCF\u0026thinsp;=\u0026thinsp;Integrated Density \u0026minus; (Area of selected cell \u0026times; Mean background fluorescence).\u003c/p\u003e\n\u003ch3\u003eIn Vivo Model of Cardiac Hypertrophy\u003c/h3\u003e\n\u003cp\u003e All animal procedures were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee of the Akdeniz University Experimental Animal Research Center (Ethics Committee Approval No: 6; Date: 05.02.2024; Protocol No: 1677/2024.02.003). One-month-old male albino Wistar rats (180\u0026ndash;240 g) were housed in stainless steel cages (four rats per cage) under controlled environmental conditions: 20\u0026ndash;24\u0026deg;C ambient temperature, 40\u0026ndash;60% relative humidity, and a 12-hour light/dark cycle. Animals had free access to standard rodent chow and water throughout the study. Rats were randomly assigned to one of five groups (n\u0026thinsp;=\u0026thinsp;8 per group): (1) Control, (2) Sham-operated (Sham), (3) AA (4) Hypertensive HCM model (HCM), and (5) HCM treated with AA (HCM\u0026thinsp;+\u0026thinsp;AA). The HCM model was induced using the two-kidney, one-clip (2K1C) method, a well-established technique that promotes cardiac remodelling through activation of the renin\u0026ndash;angiotensin\u0026ndash;aldosterone system (RAAS) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The procedure was performed as previously described [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Briefly, rats were anesthetized via intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). Following shaving and disinfection of the abdominal area with povidone-iodine, a midline laparotomy was performed. The left kidney was gently exteriorized, and the left renal artery was carefully isolated. A non-absorbable 5/0 silk suture (Boz Tıbbi Malzemeler San. Tic. A.Ş., Ankara, Turkey), coated with silicone, was placed around the renal artery using a 0.22 mm diameter guide to achieve partial occlusion. In the Sham group, the same surgical steps were performed without arterial constriction. Upon completion of the surgical procedure, the peritoneum was sutured with a 3/0 absorbable multifilament glycolic acid suture, and the skin was closed using a non-absorbable 5/0 silk suture coated with silicone (Boz Tıbbi Malzemeler San. Tic. A.Ş., Ankara, Turkey). To verify the establishment of hypertension in the 2K1C-induced groups, systolic and diastolic blood pressures (DBP) were measured weekly using a non-invasive tail-cuff system (MAY-BPHR 9610-PC; BIOPAC Systems, Santa Barbara, CA, USA). For each rat, three consecutive readings were obtained, and the average values were calculated. Measurements were conducted between 09:00 and 12:00 to minimize the influence of circadian variations. The hypertensive state was monitored over a 4-week period to confirm successful model induction.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAurantiamide Acetate Administration Protocol\u003c/h2\u003e\u003cp\u003eA stock solution of AA was prepared at a concentration of 100 mM by dissolving 10 mg of the compound in 225 \u0026micro;L of DMSO. For each rat, 2.5 \u0026micro;L of this stock solution\u0026mdash;corresponding to the appropriate dose for a 200 g animal\u0026mdash;was diluted in DMSO to a final volume of 100 \u0026micro;L and administered via intraperitoneal (i.p.) injection. The treatment regimen consisted of daily administration at a dose of 0.5 mg/kg, five days per week (totaling 2.5 mg/kg/week), for four consecutive weeks. The total cumulative dose of AA administered per animal over the treatment period was 10 mg/kg. To control for potential vehicle effects, animals in the control, sham-operated, and HCM-only groups received equivalent volumes (100 \u0026micro;L) of DMSO via i.p. injection following the same dosing schedule. At the conclusion of the four-week treatment period, animals were anesthetized with an intraperitoneal injection of ketamine (45 mg/kg) and xylazine (10 mg/kg), and subsequently euthanized. Hearts were rapidly excised. For histological evaluations, longitudinal cardiac sections were collected from four animals per group and fixed in 10% neutral-buffered formalin for hematoxylin-eosin (H\u0026amp;E), Masson\u0026rsquo;s trichrome, and immunohistochemical staining. For biochemical analyses, additional cardiac tissues were promptly snap-frozen in liquid nitrogen and stored for subsequent processing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of Cardiac Hypertrophy by Hematoxylin and Eosin Staining\u003c/h2\u003e\u003cp\u003eCardiac tissues were fixed, embedded in paraffin, and sectioned at a thickness of 4\u0026ndash;5 \u0026micro;m using a microtome. To assess morphological alterations indicative of cardiac hypertrophy, the tissue sections were stained with H\u0026amp;E following standard histological protocols [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Stained sections were examined under a Zeiss Primo Star light microscope (Carl Zeiss Microscopy GmbH, G\u0026ouml;ttingen, Germany) at 40\u0026times; magnification. High-resolution images were captured, and the cross-sectional area (CSA) of individual cardiomyocytes was quantified using Fiji software (ImageJ, version 1.54p; National Institutes of Health, Bethesda, MD, USA). Cell boundaries were manually delineated, and the average cardiomyocyte surface area (\u0026micro;m\u0026sup2;) was calculated from multiple fields per sample to ensure representative measurements.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative Assessment of Myocardial Fibrosis Using Masson\u0026rsquo;s Trichrome Staining\u003c/h2\u003e\u003cp\u003eMasson\u0026rsquo;s Trichrome staining was employed to evaluate fibrotic changes in the left ventricular myocardium [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Fixed heart tissues were embedded in paraffin and sectioned at a thickness of 5 \u0026micro;m. The sections were deparaffinized and rehydrated through a graded series of xylene and ethanol solutions. Staining was performed manually according to established protocols described in the literature [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In the stained sections, muscle fibers appeared purple, collagen fibers blue-green, and nuclei were stained dark blue to black. Representative fields from each experimental group were randomly selected, and images were captured at 40\u0026times; magnification using a Zeiss Primo Star light microscope (Carl Zeiss Microscopy GmbH, G\u0026ouml;ttingen, Germany). Fibrosis was quantified by calculating the collagen volume fraction (CVF). Image analysis was conducted using Fiji software (ImageJ version 1.54p; National Institutes of Health, Bethesda, MD, USA). The images were subjected to color deconvolution using the \u0026ldquo;Color \u0026rarr; Color Deconvolution \u0026rarr; Masson Trichrome\u0026rdquo; function to separate staining channels. Thresholding was applied specifically to the aniline blue channel, corresponding to collagen deposition. The percentage of the collagen-positive area was then calculated, and CVF was determined using the following formula: CVF (%) = (Collagen Area / Total Tissue Area) \u0026times; 100.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemical Staining of Cardiac Tissue\u003c/h2\u003e\u003cp\u003eLongitudinal sections of cardiac tissue were used for histological assessment and immunohistochemical analysis of target protein expression. Following fixation, the tissues were rinsed with distilled water, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Serial sections of 4 \u0026micro;m thickness were obtained using a microtome and mounted onto poly-L-lysine-coated glass slides. The sections were incubated overnight at 45\u0026deg;C, followed by an additional hour at 60\u0026deg;C to ensure adhesion and optimal antigen preservation prior to staining. For deparaffinization, slides were immersed in xylene twice for 20 minutes each. Rehydration was subsequently performed through descending concentrations of ethanol (100%, 90%, 80%, and 70%) for 5 minutes per step. Slides were then rinsed in distilled water and PBS (pH 7.4) for 5 minutes each. Immunohistochemical staining was conducted using the Dako Omnis automated staining system (Agilent Technologies, Santa Clara, CA, USA). Primary antibodies were diluted in PBS containing 1% bovine serum albumin (BSA) and applied at room temperature (25\u0026deg;C) for 60 minutes. The following primary antibodies were used: Polyclonal rabbit anti-GRP78 (1:100; Abcam, ab21685), Anti-p38 MAPK (1:50; Affinity Biosciences, AF6456), Anti-phospho-p38 MAPK (Thr180/Tyr182) (1:50; Affinity Biosciences, AF4001), Anti-ERK1/2 (1:100; Affinity Biosciences, AF0155), Anti-phospho-ERK1/2 (Thr202/Tyr204) (1:50; Affinity Biosciences, AF1015), Anti-JNK1/2/3 (1:50; Affinity Biosciences, AF6319), Anti-phospho-JNK1/2/3 (Thr183\u0026thinsp;+\u0026thinsp;Tyr185) (1:100; Affinity Biosciences, AF3318), Secondary detection was performed using biotinylated goat anti-rabbit IgG, followed by streptavidin-conjugated peroxidase. Immunoreactivity was visualized using 3,3\u0026prime;-diaminobenzidine (DAB) substrate, resulting in a brown precipitate indicative of positive staining. Negative controls were processed using the same protocol, substituting 5% NGS for the primary antibody. Microscopic imaging was performed using a Zeiss Primo Star light microscope at 40\u0026times; magnification. Quantitative analysis of immunostaining was carried out using Fiji (ImageJ, version 1.54p). Color deconvolution was applied via the \"Color \u0026rarr; Color Deconvolution \u0026rarr; H-DAB\" function to isolate the DAB channel. A consistent threshold for positive signal detection was manually set and applied across all images. The mean gray value of positively stained regions was measured, and optical density (OD) was calculated using the following formula: OD\u0026thinsp;=\u0026thinsp;log₁₀(255 / mean gray value). OD values from the control group were designated as 100% to represent baseline staining intensity, and all experimental groups were normalized accordingly as percentage values relative to the control. All data were subjected to statistical analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e\u003cp\u003eSerum concentrations of Ang II were quantified using a commercially available sandwich ELISA kit (BT-Laboratory, Cat. No: E0655Ra, Zhejiang, China), following the manufacturer's protocol. Diluted serum samples were applied to microplate wells pre-coated with anti-Ang II antibodies. After incubation, streptavidin-conjugated horseradish peroxidase (HRP) was added, followed by a chromogenic substrate to facilitate colour development. The enzymatic reaction was terminated using a stop solution, and absorbance was measured at 450 nm using a microplate spectrophotometer. A standard calibration curve was generated for each assay, and Ang II concentrations were calculated based on this curve. Final results were expressed in pmol/L.\u003c/p\u003e\u003cp\u003eThe expression levels of ANP, GRP78, p38 MAPK, ERK, and JNK were determined in whole heart tissue homogenates and H9C2 cell lysates using specific sandwich ELISA kits (BT-Laboratory, Zhejiang, China; ANP: Cat. No. E0642Ra; GRP78: E1255Ra; p38: E2455Ra; ERK: E1090Ra; JNK: E0048Ra). For tissue preparation, heart samples were rinsed with cold PBS and homogenized in PBS at a 1:9 (w/v) ratio. Homogenates were centrifuged at 5000 \u0026times; g for 5 minutes at 4\u0026deg;C, and the supernatants were collected for analysis. H9C2 cells were harvested according to experimental group, washed with PBS, and lysed by repeated freeze-thaw cycles followed by sonication. Each assay was performed in accordance with the respective manufacturer\u0026rsquo;s instructions. Samples were added to wells precoated with specific capture antibodies and incubated with biotinylated detection antibodies. Following the application of streptavidin-HRP and chromogenic substrate, absorbance was measured at 450 nm. Standard curves were constructed for each protein, and corresponding concentrations were extrapolated accordingly. Total protein concentrations in each sample were measured using a standard protein assay, and ELISA results were normalized to the total protein content.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eProtein Quantification\u003c/h2\u003e\u003cp\u003eTotal protein concentrations in whole heart tissue homogenates and H9C2 cell lysates were determined using a Coomassie Plus Protein Assay Kit (Pierce Chemical Company, Rockford, IL, USA), with absorbance measured at 595 nm. A standard calibration curve was generated using serial dilutions of BSA, and sample protein concentrations were calculated based on this curve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were conducted using GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, CA, USA). The specific statistical tests applied for each dataset are detailed in the corresponding figure legends. Data distributions were first assessed for normality. For datasets that did not meet the assumption of normality, appropriate non-parametric tests were utilized. Comparisons among multiple groups were performed using either one-way analysis of variance (ANOVA) or the Kruskal\u0026ndash;Wallis test, depending on data distribution. When overall significance was detected, appropriate post hoc tests were applied to determine pairwise differences between groups. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e1. Assessment of Cell Viability and Cellular Cardiac Hypertrophy\u003c/h2\u003e\u003cp\u003eThe effects of Ang II, AA, and LOS on cell viability and cardiac hypertrophy in H9C2 cells were evaluated using the MTT assay, F-actin staining, and measurements of ANP and total protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A dose-dependent cytotoxicity analysis of Ang II (0.01, 0.1, 0.2, 1, and 10 \u0026micro;M) was conducted after 24 hours of incubation using the MTT assay. No statistically significant differences in cell viability were observed among the groups (p\u0026thinsp;=\u0026thinsp;0.3034) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To determine the cytotoxic potential of AA, cells were treated with increasing concentrations (3.125\u0026ndash;100 \u0026micro;M) for 24 hours. While no cytotoxicity was detected at low to moderate doses, the highest concentration (100 \u0026micro;M) significantly reduced cell viability compared to all other doses (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The cytotoxic effects of Los were assessed at 5 and 10 \u0026micro;M following a 25-hour incubation. No significant differences in viability were observed between these groups (p\u0026thinsp;=\u0026thinsp;0.2392) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To evaluate potential interactions, DMSO (1 \u0026micro;L/mL), Ang II (1 \u0026micro;M), AA (6.25 \u0026micro;M), and Los (10 \u0026micro;M) were administered individually or in combination. No significant differences in cell viability were observed among the groups (p\u0026thinsp;=\u0026thinsp;0.1911) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These results indicate that the selected concentrations of these agents did not exhibit cytotoxic effects. To assess morphological changes associated with hypertrophy, F-actin was stained with phalloidin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Control cells displayed normal morphology with well-defined borders, whereas Ang II-treated cells exhibited marked enlargement. Treatment with AA and/or Los visually attenuated the Ang II-induced morphological changes. These observations were corroborated by quantitative cell surface area analysis. Compared to controls, Ang II significantly increased cell surface area (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), which was markedly reduced by AA and Los treatments. Notably, in the Ang II\u0026thinsp;+\u0026thinsp;AA and Los\u0026thinsp;+\u0026thinsp;Ang II\u0026thinsp;+\u0026thinsp;AA groups, surface area values approached those of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). ANP protein levels, a molecular marker of cardiac hypertrophy, were measured in cell lysates using ELISA. ANP expression was significantly elevated in the Ang II group, whereas co-treatment with losartan, AA, and Ang II significantly reduced ANP levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Analysis of total protein content revealed the highest levels in the Ang II-treated group. This increase was significantly attenuated by AA, LOS, and their combination therapies (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2. Effects of Endoplasmic Reticulum Stress on Cellular Cardiac Hypertrophy\u003c/h2\u003e\u003cp\u003eThe involvement of ER stress in cardiac hypertrophy and the efficacy of therapeutic interventions targeting this stress were investigated in H9C2 cells. Key parameters assessed included cell viability, GRP78 expression levels, F-actin staining for morphological analysis, and total protein content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A model of ER stress was established using TM, and the modulatory effects of TUDCA and AA were systematically evaluated. The cytotoxic effects of TM were assessed via MTT assay following 24-hour incubation across a concentration range of 0.5\u0026ndash;10 \u0026micro;g/mL. TM treatment at 1, 2, 5, and 10 \u0026micro;g/mL significantly reduced cell viability compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while 0.5 \u0026micro;g/mL did not result in a statistically significant cytotoxic effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). TUDCA, a known ER stress inhibitor, was evaluated at doses ranging from 0.06\u0026ndash;4 mM over 24 hours. While low to moderate concentrations were well tolerated, the highest dose (4 mM) significantly decreased cell viability (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Based on these findings, 1 mM TUDCA\u0026mdash;identified as the highest non-cytotoxic concentration\u0026mdash;was selected for subsequent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To examine the combined effects of various agents, DMSO (1 \u0026micro;L/mL), TM (0.5 \u0026micro;g/mL), Ang II (1 \u0026micro;M), TUDCA (1 mM), and AA (6.25 \u0026micro;M) were applied individually and in combination. A significant reduction in cell viability was observed only in the group receiving TM, Ang II, and AA concurrently, with viability declining to approximately 74% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant viability changes were noted in the other treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). GRP78, a molecular marker of ER stress, was assessed using both immunofluorescence (IF) staining and ELISA (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;F). IF analysis revealed that TM and Ang II significantly elevated GRP78 expression, with the TM\u0026thinsp;+\u0026thinsp;Ang II group showing the most pronounced increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Co-treatment with either TUDCA or AA markedly attenuated this elevation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). ELISA data obtained from cell lysates corroborated the IF results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Morphological changes associated with hypertrophy were visualized via phalloidin-based F-actin staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Quantitative analysis demonstrated a significant reduction in cell surface area in the TM-treated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas no significant morphological alterations were observed in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Total protein analysis showed significantly elevated levels in both TM and TM\u0026thinsp;+\u0026thinsp;Ang II groups compared to control cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). This increase was significantly suppressed by TUDCA and AA co-treatments (TM\u0026thinsp;+\u0026thinsp;TUDCA, TM\u0026thinsp;+\u0026thinsp;AA) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Notably, protein levels in the TM\u0026thinsp;+\u0026thinsp;Ang II\u0026thinsp;+\u0026thinsp;AA group were further reduced compared to those in the TM\u0026thinsp;+\u0026thinsp;Ang II\u0026thinsp;+\u0026thinsp;TUDCA group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Collectively, these findings indicate that ER stress-induced protein accumulation in H9C2 cells can be mitigated by both AA and TUDCA, with AA demonstrating a potentially greater protective effect. These results support the therapeutic potential of AA in conditions characterized by ER stress-associated cardiac hypertrophy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3. In Vivo Evaluation of Ang II and Endoplasmic Reticulum Stress in Hypertensive Hypertrophic Cardiomyopathy\u003c/h2\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.1. Blood Pressure Changes and Ang II Levels\u003c/h2\u003e\u003cp\u003eIn a HCM model, changes in systolic and DBP, as well as serum Ang II levels, were assessed over a 4-week period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;C). Baseline systolic blood pressure (SBP) was comparable across all groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), with no significant difference observed between the control and sham groups throughout the study (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the HCM group, SBP increased significantly from the first week (*, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). A similar increase was observed in the HCM\u0026thinsp;+\u0026thinsp;AA group during the first week (*, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01); however, SBP gradually declined in subsequent weeks and returned to baseline by week four. SBP in the HCM group was significantly higher than in the control, sham, and AA groups at all time points (#, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the HCM\u0026thinsp;+\u0026thinsp;AA group, SBP was elevated during the first three weeks compared to the control groups (#, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but showed no significant difference in the fourth week (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). From the second week onward, SBP in the HCM\u0026thinsp;+\u0026thinsp;AA group was significantly lower than in the untreated HCM group (\u0026sect;, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, SBP in the AA group was significantly lower than in the control at week two (\u0026para;, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Diastolic blood pressure followed a similar trend. Initial DBP values were comparable across all groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), with no significant differences between control and sham groups during the study period (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). DBP in the HCM group increased significantly from the first week onward (*, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and a similar pattern was observed in the HCM\u0026thinsp;+\u0026thinsp;AA group during the first week (*, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, DBP declined in later weeks and returned to baseline by week four. DBP was significantly higher in the HCM group compared to control, sham, and AA groups at all time points (#, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the HCM\u0026thinsp;+\u0026thinsp;AA group, DBP was higher during the first three weeks (#, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but not in week four (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Notably, DBP in the HCM\u0026thinsp;+\u0026thinsp;AA group decreased significantly from the second week, with values lower than those in the HCM group (\u0026sect;, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At week three, DBP in the AA group was significantly lower than both the HCM\u0026thinsp;+\u0026thinsp;AA and control groups (\u0026para;, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eSerum Ang II levels, assessed by ELISA at the end of week four, were significantly elevated in the HCM group compared to all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating systemic activation of the RAAS in this renovascular hypertension model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). AA treatment markedly reduced this elevation in the HCM\u0026thinsp;+\u0026thinsp;AA group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e3.2. Modeling Hypertrophic Cardiomyopathy In Vivo\u003c/h2\u003e\u003cp\u003eHistological and biochemical assessments were performed on left ventricular myocardium to evaluate hypertrophic remodelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;G). H\u0026amp;E staining of longitudinal heart sections revealed a marked increase in cardiomyocyte size in the HCM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), which was confirmed quantitatively by cross-sectional area analysis. Average cardiomyocyte cross-sectional area in the HCM group was significantly higher than in all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and AA treatment significantly reduced this hypertrophic response (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eANP, a molecular marker of cardiac hypertrophy, was assessed by ELISA. ANP levels were significantly elevated in the HCM group compared to all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas AA treatment reduced ANP expression to near-control levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Similarly, total cardiac protein concentration was significantly elevated in the HCM group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while AA treatment suppressed this increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Fibrosis Analysis\u003c/h2\u003e\u003cp\u003eCardiac fibrosis was assessed by Masson's trichrome staining of left ventricular myocardium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH\u0026ndash;I). Prominent collagen accumulation was observed in the HCM group (black arrows), while control, sham, and AA groups exhibited a more organized myocardial structure with minimal fibrotic areas. In the HCM\u0026thinsp;+\u0026thinsp;AA group, fibrosis was markedly reduced, and tissue architecture appeared more preserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Collagen volume fraction quantified using ImageJ, was significantly higher in the HCM group (50.07\u0026thinsp;\u0026plusmn;\u0026thinsp;6.26%) compared to control (18.54\u0026thinsp;\u0026plusmn;\u0026thinsp;6.93%), sham (16.44\u0026thinsp;\u0026plusmn;\u0026thinsp;8.71%), AA (19.57\u0026thinsp;\u0026plusmn;\u0026thinsp;4.23%), and HCM\u0026thinsp;+\u0026thinsp;AA (25.48\u0026thinsp;\u0026plusmn;\u0026thinsp;4.04%) groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). AA treatment significantly attenuated collagen accumulation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e3.4. Cardiac Hypertrophy Assessment\u003c/h2\u003e\u003cp\u003eAbsolute heart weight and heart weight-to-body weight ratio (HW/BW) were measured to assess cardiac remodelling. Heart weight was significantly higher in the HCM group (936.5\u0026thinsp;\u0026plusmn;\u0026thinsp;39.00 mg) than in the control (734.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.64 mg), sham (738.0\u0026thinsp;\u0026plusmn;\u0026thinsp;37.71 mg), AA (685.3\u0026thinsp;\u0026plusmn;\u0026thinsp;23.84 mg), and HCM\u0026thinsp;+\u0026thinsp;AA (802.0\u0026thinsp;\u0026plusmn;\u0026thinsp;32.02 mg) groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). AA treatment significantly suppressed the HCM-induced increase in heart weight (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Similarly, HW/BW ratio was significantly elevated in the HCM group (3.725\u0026thinsp;\u0026plusmn;\u0026thinsp;0.379) compared to control (2.897\u0026thinsp;\u0026plusmn;\u0026thinsp;0.449), sham (2.797\u0026thinsp;\u0026plusmn;\u0026thinsp;0.130), and AA (2.854\u0026thinsp;\u0026plusmn;\u0026thinsp;0.340) groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and was significantly reduced in the HCM\u0026thinsp;+\u0026thinsp;AA group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). These findings demonstrate that AA mitigates cardiac hypertrophy and relative cardiac loading associated with hypertensive remodelling.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e3.5. In Vivo Assessment of Endoplasmic Reticulum Stress\u003c/h2\u003e\u003cp\u003eTo evaluate ER stress in hypertensive HCM, GRP78 protein expression in left ventricular myocardium was analysed via immunohistochemistry (IHC) and ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL\u0026ndash;N). IHC staining revealed a significant increase in GRP78-positive cells and DAB staining intensity in the HCM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Optical density analysis confirmed significantly higher GRP78 immunoreactivity in the HCM group compared to control, sham, AA, and HCM\u0026thinsp;+\u0026thinsp;AA groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). ELISA results supported these findings, showing significantly elevated GRP78 levels in the HCM group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while AA treatment reduced GRP78 expression in the HCM\u0026thinsp;+\u0026thinsp;AA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN). These results indicate that AA effectively alleviates ER stress induced by hypertensive cardiac remodelling in vivo.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e4. In Vitro Evaluation of MAPK Signalling Pathway Components in H9C2 Cells\u003c/h2\u003e\u003cp\u003eTo assess the activation of the MAPK signalling pathway in cellular hypertrophy, the total and phosphorylated forms of p38 MAPK, ERK, and JNK were examined in H9C2 cardiomyoblasts using immunofluorescence staining and ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Immunofluorescence analysis of p38 and p-p38 MAPK showed no discernible difference in the fluorescence intensity of total p38 MAPK among the experimental groups. In contrast, p-p38 MAPK fluorescence intensity was markedly elevated in cells treated with Ang II, while treatment with AA, Los, or their combination significantly diminished this increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Quantitative analyses by both immunofluorescence and ELISA confirmed that total p38 MAPK levels remained unchanged across all groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;C). However, p-p38 MAPK levels were significantly increased in the Ang II group and were effectively suppressed by AA and/or Los treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Consistent with these findings, the p-p38/p38 ratio was highest in the Ang II group and was significantly reduced by AA and losartan treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). For ERK, neither immunofluorescence nor ELISA revealed significant differences in total ERK expression among the groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG\u0026ndash;H). However, p-ERK levels were significantly elevated in Ang II-treated cells and were substantially attenuated by AA, Los, or combination therapy (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). The p-ERK/ERK ratio was also significantly increased in the Ang II group and normalized following AA and/or Los treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Similar trends were observed for JNK. Total JNK expression did not significantly differ between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL\u0026ndash;M). Nevertheless, p-JNK fluorescence intensity was significantly increased in the Ang II group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and this effect was significantly mitigated by AA and/or Los p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN). Analysis of the p-JNK/JNK ratio revealed a marked increase in the Ang II group compared to the control, Ang II\u0026thinsp;+\u0026thinsp;AA, Los\u0026thinsp;+\u0026thinsp;Ang II, and Los\u0026thinsp;+\u0026thinsp;Ang II\u0026thinsp;+\u0026thinsp;AA groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO). Collectively, these results demonstrate that Ang II activates the MAPK signalling pathway by enhancing the phosphorylation of p38, ERK, and JNK. AA significantly suppresses this activation, underscoring its potential as a modulator of MAPK-mediated hypertrophic signalling.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e5. In Vivo Assessment of MAPK Signalling Pathway Activation\u003c/h2\u003e\u003cp\u003eTo evaluate the activation status of the MAPK signaling cascade in vivo, immunohistochemical staining and ELISA were employed to assess the total and phosphorylated forms of p38 MAPK, ERK, and JNK in left ventricular myocardium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Immunohistochemical analysis showed no significant differences in total p38 MAPK staining intensity among the experimental groups, a finding corroborated by ELISA quantification in total heart tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;C). However, p-p38 MAPK levels were significantly elevated in the HCM group compared to all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), as determined by image analysis of staining intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Furthermore, the ratio of p-p38 MAPK to total p38 MAPK was also significantly increased in the HCM group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating activation of the p38 MAPK pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Similarly, no significant differences were detected in total ERK expression across groups by either IHC or ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;H). In contrast, p-ERK staining intensity was markedly higher in the HCM group compared to all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and this elevation was reflected in the p-ERK/ERK ratio, which was significantly increased in the HCM group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI\u0026ndash;J). These results suggest that ERK phosphorylation is a prominent feature of MAPK pathway activation in the hypertrophic myocardium. Total JNK levels showed no significant variation among groups based on both IHC and ELISA analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK\u0026ndash;M). However, p-JNK levels were significantly increased in the HCM group compared to all other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as determined by staining intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). Correspondingly, the p-JNK/JNK ratio was significantly elevated in the HCM group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), further confirming activation of the JNK arm of the MAPK signalling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO). Collectively, these data demonstrate that while total protein levels of p38 MAPK, ERK, and JNK remain unchanged in hypertrophic myocardium, phosphorylation of these kinases is significantly upregulated. These findings confirm the activation of the MAPK signalling cascade, particularly in response to Ang II-mediated hypertrophic stimuli in vivo.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHypertensive cardiac hypertrophy represents a major pathological adaptation to increased afterload, frequently progressing to heart failure if left unchecked [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The hypertrophic process is characterized by cardiomyocyte enlargement, fibrosis, and maladaptive remodelling, leading to compromised cardiac function [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Our study demonstrates for the first time that AA, a dipeptide derivative found in edible plants and marine fungi, mitigates Ang II-induced hypertrophy and ER stress both in vitro and in a 2K1C rat model. These findings underscore the cardioprotective potential of AA via modulation of MAPK signaling and ER stress pathways, providing novel insights into its therapeutic application against hypertensive heart disease.\u003c/p\u003e\u003cp\u003eCardiac hypertrophy is a compensatory mechanism in response to elevated hemodynamic stress, prominently mediated by Ang II signaling [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Ang II promotes hypertrophic growth through AT1R-dependent activation of downstream signaling cascades, notably the MAPK pathway, resulting in pathological remodelling [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Consistent with previous findings, our study revealed that Ang II markedly increased H9C2 cell size, ANP levels, and total protein content, confirming successful hypertrophic induction. For instance, a study investigated the effects of Ang II on H9C2 cells and found that treatment with 1 \u0026micro;mol/L Ang II for 24 hours resulted in marked increases in cell surface area, total protein synthesis, and mRNA expression levels of hypertrophic markers such as ANP and brain natriuretic peptide (BNP) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Treatment with AA significantly attenuated Ang II-induced hypertrophy, reducing both cellular surface area and ANP expression. Total protein content, another hallmark of hypertrophic remodelling, was also significantly reduced following AA treatment. The suppression of ANP expression is critical, as elevated ANP levels are a compensatory response to hemodynamic stress but are indicative of pathological remodelling [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These findings are consistent with the anti-inflammatory and vasoprotective effects of AA reported in hypertensive models [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], suggesting a direct anti-hypertrophic role.\u003c/p\u003e\u003cp\u003eThe 2K1C model recapitulates human renovascular hypertension and its associated cardiac remodelling [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Consistent with previous reports, rats subjected to 2K1C developed marked hypertension, cardiac hypertrophy, fibrosis, and elevated Ang II levels. Our findings revealed that AA administration ameliorated these pathological changes, significantly lowering SBP and DBP. Notably, serum Ang II concentrations were markedly reduced in AA-treated rats, suggesting an upstream modulation of the RAAS system. This reduction in Ang II levels is significant because Ang II not only promotes hypertension but also induces oxidative stress, inflammation, and fibrosis, contributing to the progression of cardiac hypertrophy [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Histological analysis demonstrated decreased cardiomyocyte cross-sectional area and reduced collagen deposition in AA-treated hearts, indicative of reduced hypertrophy and fibrosis. Given that fibrosis stiffens the myocardium and impairs diastolic function [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the anti-fibrotic effect of AA suggests potential benefits in preventing heart failure progression. The reduction in heart weight and heart weight-to-body weight ratios in AA-treated animals corroborates the anti-hypertrophic efficacy observed histologically. The observed decrease in total myocardial protein content, as well as lowered ANP levels, reinforces the anti-remodelling effect of AA. ANP, a marker of myocardial stress, is upregulated in response to volume overload and hypertrophy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]; thus, its normalization by AA treatment reflects a reversal of pathological remodelling.\u003c/p\u003e\u003cp\u003eActivation of MAPK pathways, including p38, ERK1/2, and JNK, plays a pivotal role in pathological hypertrophic responses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Our results demonstrated that Ang II stimulation increased phosphorylation of p38, ERK, and JNK in H9C2 cells without affecting total protein levels. This selective upregulation of phosphorylated MAPKs aligns with previous observations that MAPK activation, rather than expression, is key to hypertrophic signaling [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. AA treatment significantly reduced phosphorylation levels of all three MAPK branches, restoring them close to baseline. The inhibition of MAPK phosphorylation by AA is critical because these kinases regulate transcription factors such as NF-κB and AP-1, which are responsible for the expression of pro-hypertrophic genes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. By modulating these signalling cascades, AA effectively prevents the transcriptional upregulation of hypertrophic genes. Importantly, the inhibition of MAPK signaling pathways in vivo corroborates findings in other models of cardiac hypertrophy where blockade of MAPK cascades attenuated hypertrophy and improved cardiac function [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eER stress, resulting from the accumulation of unfolded proteins, is a well-documented contributor to cardiac hypertrophy and failure [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In our in vitro model, TM-induced ER stress increased GRP78 expression, a hallmark of the UPR, which was further exacerbated by Ang II. Importantly, AA significantly reduced GRP78 levels, comparable to TUDCA, a known ER stress inhibitor. These findings suggest that AA not only attenuates hypertrophy via MAPK inhibition but also mitigates ER stress, potentially by enhancing protein folding capacity or facilitating proteostasis. Previous studies have shown that prolonged ER stress leads to activation of pro-apoptotic pathways, such as CHOP-mediated apoptosis, contributing to cardiomyocyte loss and cardiac dysfunction [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Morphological analysis confirmed that ER stress exacerbates hypertrophic changes, which were alleviated by AA treatment. The ability of AA to modulate ER stress pathways positions it as a unique therapeutic candidate targeting multiple hypertrophic mechanisms simultaneously. Moreover, the reduction in ER stress markers suggests that AA may preserve protein homeostasis, thereby protecting cardiomyocytes from stress-induced apoptosis and maladaptive remodelling.\u003c/p\u003e\u003cp\u003eCurrent anti-hypertrophic therapies, such as RAAS inhibitors, beta-blockers, and mineralocorticoid receptor antagonists, primarily target neurohormonal activation but do not directly modulate intracellular stress responses like ER stress [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although effective in reducing mortality, these therapies often fail to completely reverse structural cardiac changes once hypertrophy is established. AA's dual-action profile offers a potential therapeutic advantage by concurrently targeting upstream and downstream pathological processes. By mitigating both MAPK pathway activation and ER stress, AA may halt the feed-forward cycle of hypertrophy and fibrosis more effectively than current single-target therapies. This multi-targeted approach is particularly advantageous in complex diseases such as hypertensive heart disease, where multiple signalling pathways are dysregulated. The precise molecular targets of AA remain to be elucidated. Its structure, comprising N-benzoylphenylalanine and phenylalanine residues [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], suggests potential interactions with intracellular signaling molecules or chaperones involved in the UPR and MAPK cascades. Proteomic and transcriptomic studies could elucidate these interactions and identify key mediators of AA\u0026rsquo;s effects. Furthermore, additional investigations into AA's effects on oxidative stress and autophagy pathways, both of which are implicated in hypertensive cardiac remodelling, could provide a more comprehensive understanding of its cardioprotective mechanisms [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Future studies should also focus on dose-response relationships, long-term efficacy, and safety profiles in larger animal models. Given the chronic nature of hypertension and its associated cardiac complications, it is imperative to assess the long-term effects of AA treatment on cardiac structure and function. Additionally, studies investigating the combination of AA with existing therapies such as RAAS inhibitors could reveal synergistic effects, offering new avenues for combination therapy in hypertensive heart disease.\u003c/p\u003e\u003cp\u003eAn important aspect of our findings is the distinction between the mechanisms observed in vitro versus in vivo. In cell-based experiments, AA treatment appears to block Ang II signaling at the receptor or post-receptor level, inhibiting downstream MAPK activation and ER stress. In contrast, in the in vivo model, AA leads to a significant reduction in circulating Ang II levels, suggesting an effect on the synthesis or release of Ang II itself. At first glance, these may seem like two separate phenomena\u0026mdash;one targeting receptor-mediated signaling, the other modulating systemic RAAS activity. However, we propose that these actions are not contradictory but rather reflect a cohesive, multi-level mechanism of AA\u0026rsquo;s cardioprotective effects. AA may exert local effects within cardiomyocytes while simultaneously influencing systemic regulators of Ang II production through vascular or renal pathways. This integrated mode of action provides a more complete understanding of how AA mitigates hypertensive cardiac remodeling, unifying both cellular and systemic effects into a single, biologically plausible therapeutic narrative. Several pharmacological agents and natural compounds exhibit dual actions similar to AA, targeting both systemic RAAS activity and intracellular hypertrophic signaling. ARBs like losartan and ACE inhibitors reduce Ang II levels in vivo while inhibiting MAPK pathways in vitro [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Natural compounds such as resveratrol and berberine lower blood pressure and Ang II, while also blocking Ang II-induced hypertrophy and ER stress in cardiomyocytes [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven the increasing global burden of hypertension-induced cardiac hypertrophy and the limited efficacy of existing therapies in reversing structural cardiac changes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], AA represents a promising candidate for adjunctive therapy. Its ability to modulate multiple pathological pathways simultaneously could translate into superior therapeutic outcomes for patients with hypertensive heart disease. Moreover, the natural origin of AA, found in edible plants and marine-derived fungi, may offer an advantage in terms of biocompatibility and reduced side effect profiles compared to synthetic pharmacological agents. However, comprehensive toxicological evaluations are necessary to confirm its safety profile.\u003c/p\u003e\u003cp\u003eThis study has several limitations. First, only a single dose of AA was tested in vivo; dose-response studies are warranted to establish optimal therapeutic windows. Second, long-term outcomes and potential off-target effects of AA were not assessed. Third, molecular docking and target validation studies are needed to confirm the direct interactions of AA with MAPK or ER stress components. Additionally, the lack of direct measurement of other stress pathways such as oxidative stress or autophagy limits our understanding of AA\u0026rsquo;s full spectrum of action. Future studies should also assess the effects of AA in female animal models, as sex differences in hypertensive cardiac remodeling have been reported [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn conclusion, 0ur study provides compelling evidence that AA mitigates hypertensive cardiac hypertrophy through inhibition of MAPK signalling and ER stress both in vitro and in vivo. These findings position AA as a promising therapeutic agent warranting further investigation for the treatment of hypertensive heart disease. Comprehensive preclinical studies are necessary to validate these findings and to facilitate the clinical translation of AA for use in patients with hypertension-induced cardiac hypertrophy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAA, Aurantiamide acetate\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eANP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAtrial Natriuretic Peptide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAng II\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAngiotensin II\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAT1R\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAngiotensin II type 1 receptor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCVF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCollagen Volume Fraction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDBP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDiastolic Blood Pressure\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDimethyl sulfoxide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eeNOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEndothelial Nitric Oxide Synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEnzyme-Linked Immunosorbent Assay\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eER\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEndoplasmic Reticulum\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eERK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eExtracellular signal-regulated kinase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFetal Bovine Serum\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGRP78\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlucose-Regulated Protein 78\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eH9C2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRat Cardiomyoblast Cell Line\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHCM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHypertrophic Cardiomyopathy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eImmunohistochemistry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eJNK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ec-Jun N-terminal kinase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMAPK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMitogen-Activated Protein Kinase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMTT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNaCl\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSodium Chloride\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOptical Density\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhosphate Buffered Saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRAAS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRenin-Angiotensin-Aldosterone System\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSBP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSystolic Blood Pressure\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStandard Deviation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTunicamycin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTUDCA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTauroursodeoxycholic Acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eUPR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eUnfolded Protein Response.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics approval\u003c/h2\u003e\u003cp\u003e This study was reviewed and approved by Akdeniz University Experimental Animals Application and Research Center, Animal Experiments Local Ethics Committee (Ethics Committee Approval No: 6; Date: 05.02.2024; Protocol No: 1677/2024.02.003)\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eClinical trial number\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConsent to participate\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Akdeniz University Research Foundation (Grant No: TDK-2024-6565) and the Scientific and Technological Research Council of T\u0026uuml;rkiye (T\u0026Uuml;BİTAK) under Project No: 124S301.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe authors declare that all data were generated in-house and that no paper mill was used. T\u0026Ccedil;, carried out animal studies, investigation, validation, formal analysis, reviewing, and editing. MA, conceptualization, funding acquisition, supervision, writing, reviewing, and editing. EK, carried out histological studies. HTB, carried out pathological studies. \u0026Ccedil;Y, carried out cell culture, animal studies, immunohistochemical measurements and analysed data. A\u0026Ouml;\u0026Uuml;, carried out cell culture, animal studies, immunohistochemical measurements and analysed data.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to acknowledge Dr. Gamze Tanrı\u0026ouml;ver for her valuable advice and general guidance during the project\u003c/p\u003e\u003ch2\u003eAvailability of data and material\u003c/h2\u003e\u003cp\u003eData obtained and analysed are available from the corresponding author on reasonable request.\u003c/p\u003e\u003ch2\u003eCode availability\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMarian AJ, Braunwald E. Hypertrophic Cardiomyopathy: Genetics, Pathogenesis, Clinical Manifestations, Diagnosis, and Therapy. Circ Res. 2017 Sep 15;121(7):749-770. doi: 10.1161/CIRCRESAHA.117.311059. PMID: 28912181; PMCID: PMC5654557.\u003c/li\u003e\n\u003cli\u003eSaheera S, Krishnamurthy P. Cardiovascular Changes Associated with Hypertensive Heart Disease and Aging. Cell Transplant. 2020 Jan-Dec;29:963689720920830. doi: 10.1177/0963689720920830. PMID: 32393064; PMCID: PMC7586256.\u003c/li\u003e\n\u003cli\u003eCarter K, Shah E, Waite J, Rana D, Zhao ZQ. Pathophysiology of Angiotensin II-Mediated Hypertension, Cardiac Hypertrophy, and Failure: A Perspective from Macrophages. Cells. 2024 Dec 4;13(23):2001. doi: 10.3390/cells13232001. PMID: 39682749; PMCID: PMC11640308.\u003c/li\u003e\n\u003cli\u003eBhullar SK, Dhalla NS. Angiotensin II-Induced Signal Transduction Mechanisms for Cardiac Hypertrophy. Cells. 2022 Oct 22;11(21):3336. doi: 10.3390/cells11213336. PMID: 36359731; PMCID: PMC9657342.\u003c/li\u003e\n\u003cli\u003eDarling NJ, Cook SJ. The role of MAPK signalling pathways in the response to endoplasmic reticulum stress. Biochim Biophys Acta. 2014 Oct;1843(10):2150-63. doi: 10.1016/j.bbamcr.2014.01.009. PMID: 24440275.\u003c/li\u003e\n\u003cli\u003eAn Y, Wang X, Guan X, Yuan P, Liu Y, Wei L, Wang F, Qi X. Endoplasmic reticulum stress-mediated cell death in cardiovascular disease. Cell Stress Chaperones. 2024 Feb;29(1):158-174. doi: 10.1016/j.cstres.2023.12.003. PMID: 38295944; PMCID: PMC10939083.\u003c/li\u003e\n\u003cli\u003eRose BA, Force T, Wang Y. Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale. Physiol Rev. 2010 Oct;90(4):1507-46. doi: 10.1152/physrev.00054.2009. PMID: 20959622; PMCID: PMC3808831.\u003c/li\u003e\n\u003cli\u003eYao Y, Lu Q, Hu Z, Yu Y, Chen Q, Wang QK. A non-canonical pathway regulates ER stress signaling and blocks ER stress-induced apoptosis and heart failure. Nat Commun. 2017 Jul 25;8(1):133. doi: 10.1038/s41467-017-00171-w. PMID: 28743963; PMCID: PMC5527107.\u003c/li\u003e\n\u003cli\u003eGhorani V, Saadat S, Khazdair MR, Gholamnezhad Z, El-Seedi H, Boskabady MH. Phytochemical Characteristics and Anti-Inflammatory, Immunoregulatory, and Antioxidant Effects of Portulaca oleracea L.: A Comprehensive Review. Evid Based Complement Alternat Med. 2023 Aug 31;2023:2075444. doi: 10.1155/2023/2075444. PMID: 37693918; PMCID: PMC10484659.\u003c/li\u003e\n\u003cli\u003eSuhas R, Channe Gowda D. Structure-based rationale design and synthesis of aurantiamide acetate analogues - towards a new class of potent analgesic and anti-inflammatory agents. Chem Biol Drug Des. 2012 May;79(5):850-62. doi: 10.1111/j.1747-0285.2012.01331.x. PMID: 22251852.\u003c/li\u003e\n\u003cli\u003eAslan M, Basralı F, \u0026Uuml;lker P, Barut Z, Yılmaz \u0026Ccedil;, \u0026Ccedil;eker T, \u0026Ouml;zen N, \u0026Ouml;zt\u0026uuml;z\u0026uuml;n A, Elpek \u0026Ouml;. Effects of aurantiamide on a rat model of renovascular arterial hypertension. Pflugers Arch. 2023 Oct;475(10):1177-1192. doi: 10.1007/s00424-023-02850-8. PMID: 37582694; PMCID: PMC10499692.\u003c/li\u003e\n\u003cli\u003eHern\u0026aacute;ndez JS, Barreto-Torres G, Kuznetsov AV, Khuchua Z, Javadov S. Crosstalk between AMPK activation and angiotensin II-induced hypertrophy in cardiomyocytes: the role of mitochondria. J Cell Mol Med. 2014 Apr;18(4):709-20. doi: 10.1111/jcmm.12220. PMID: 24444314; PMCID: PMC3981893.\u003c/li\u003e\n\u003cli\u003eThimm C, Erichsen L, Wruck W, Adjaye J. Unveiling Angiotensin II and Losartan-Induced Gene Regulatory Networks Using Human Urine-Derived Podocytes. Int J Mol Sci. 2023 Jun 23;24(13):10551. doi: 10.3390/ijms241310551. PMID: 37445727; PMCID: PMC10341883.\u003c/li\u003e\n\u003cli\u003eAfşar E, Kırımlıoglu E, \u0026Ccedil;eker T, Yılmaz \u0026Ccedil;, Demir N, Aslan M. Effect of ER stress on sphingolipid levels and apoptotic pathways in retinal pigment epithelial cells. Redox Biol. 2020 Feb;30:101430. doi: 10.1016/j.redox.2020.101430. PMID: 31978676; PMCID: PMC6976939.\u003c/li\u003e\n\u003cli\u003eYang Y, Mbikyo MB, Zhang J, Zhang Y, Zhang N, Li Z. The lncRNA MIAT regulates CPT-1a mediated cardiac hypertrophy through m6A RNA methylation reading protein Ythdf2. Cell Death Discov. 2022 Apr 5;8(1):167. doi: 10.1038/s41420-022-00977-8. PMID: 35383152; PMCID: PMC8983679.\u003c/li\u003e\n\u003cli\u003eIampanichakul M, Poasakate A, Potue P, Rattanakanokchai S, Maneesai P, Prachaney P, Settheetham-Ishida W, Pakdeechote P. Nobiletin resolves left ventricular and renal changes in 2K-1C hypertensive rats. Sci Rep. 2022 Jun 3;12(1):9289. doi: 10.1038/s41598-022-13513-6. PMID: 35662276; PMCID: PMC9166784.\u003c/li\u003e\n\u003cli\u003eCooper STE, Westaby JD, Haines ZHR, Malone GO, Sheppard MN, Meijles DN. Of Mouse and Man: Cross-Species Characterization of Hypertensive Cardiac Remodeling. Int J Mol Sci. 2022 Jul 12;23(14):7709. doi: 10.3390/ijms23147709. PMID: 35887055; PMCID: PMC9323458.\u003c/li\u003e\n\u003cli\u003eDaunoravicius D, Besusparis J, Zurauskas E, Laurinaviciene A, Bironaite D, Pankuweit S, Plancoulaine B, Herlin P, Bogomolovas J, Grabauskiene V, Laurinavicius A. Quantification of myocardial fibrosis by digital image analysis and interactive stereology. Diagn Pathol. 2014 Jun 9;9:114. doi: 10.1186/1746-1596-9-114. PMID: 24912374; PMCID: PMC4072260.\u003c/li\u003e\n\u003cli\u003eGomori G. A rapid one-step trichrome stain. Am J Clin Pathol. 1950 Jul;20(7):661-4. doi: 10.1093/ajcp/20.7_ts.661. PMID: 15432364.\u003c/li\u003e\n\u003cli\u003eHan J, Li J, Liu L, Li K, Zhang C, Han Y. 20-HETE mediates Ang II-induced cardiac hypertrophy via ROS and Ca2+ signaling in H9c2 cells. Sci Rep. 2025 Jan 17;15(1):2342. doi: 10.1038/s41598-025-85992-2. PMID: 39825084; PMCID: PMC11742049.\u003c/li\u003e\n\u003cli\u003eGuan XH, Hong X, Zhao N, Liu XH, Xiao YF, Chen TT, Deng LB, Wang XL, Wang JB, Ji GJ, Fu M, Deng KY, Xin HB. CD38 promotes angiotensin II-induced cardiac hypertrophy. J Cell Mol Med. 2017 Aug;21(8):1492-1502. doi: 10.1111/jcmm.13076. PMID: 28296029; PMCID: PMC5542907.\u003c/li\u003e\n\u003cli\u003eMehta PK, Griendling KK. Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol. 2007 Jan;292(1):C82-97. doi: 10.1152/ajpcell.00287.2006. PMID: 16870827.\u003c/li\u003e\n\u003cli\u003eFrangogiannis NG. Cardiac fibrosis. Cardiovasc Res. 2021 May 25;117(6):1450-1488. doi: 10.1093/cvr/cvaa324. PMID: 33135058; PMCID: PMC8152700.\u003c/li\u003e\n\u003cli\u003eCavallero S, Gonz\u0026aacute;lez GE, Puy\u0026oacute; AM, Ros\u0026oacute;n MI, P\u0026eacute;rez S, Morales C, Hertig CM, Gelpi RJ, Fern\u0026aacute;ndez BE. Atrial natriuretic peptide behaviour and myocyte hypertrophic profile in combined pressure and volume-induced cardiac hypertrophy. J Hypertens. 2007 Sep;25(9):1940-50. doi: 10.1097/HJH.0b013e3282435b1e. PMID: 17762660.\u003c/li\u003e\n\u003cli\u003eSugden PH, Clerk A. \u0026quot;Stress-responsive\u0026quot; mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res. 1998 Aug 24;83(4):345-52. doi: 10.1161/01.res.83.4.345. PMID: 9721691.\u003c/li\u003e\n\u003cli\u003eBueno OF, Molkentin JD. Involvement of extracellular signal-regulated kinases 1/2 in cardiac hypertrophy and cell death. Circ Res. 2002 Nov 1;91(9):776-81. doi: 10.1161/01.res.0000038488.38975.1a. PMID: 12411391.\u003c/li\u003e\n\u003cli\u003eSzegezdi E, Logue SE, Gorman AM, Samali A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006 Sep;7(9):880-5. doi: 10.1038/sj.embor.7400779. PMID: 16953201; PMCID: PMC1559676.\u003c/li\u003e\n\u003cli\u003eKittleson MM. A Clinician\u0026apos;s Guide to the 2022 ACC/AHA/HFSA Guideline for the Management of Heart Failure. J Card Fail. 2022 May;28(5):831-834. doi: 10.1016/j.cardfail.2022.03.346. PMID: 35378258.\u003c/li\u003e\n\u003cli\u003eNishida K, Otsu K. Autophagy during cardiac remodeling. J Mol Cell Cardiol. 2016 Jun;95:11-8. doi: 10.1016/j.yjmcc.2015.12.003. Epub 2015 Dec 8. PMID: 26678624.\u003c/li\u003e\n\u003cli\u003eFerrario CM, Mullick AE. Renin angiotensin aldosterone inhibition in the treatment of cardiovascular disease. Pharmacol Res. 2017 Nov;125(Pt A):57-71. doi: 10.1016/j.phrs.2017.05.020. Epub 2017 May 29. PMID: 28571891; PMCID: PMC5648016.\u003c/li\u003e\n\u003cli\u003eMa E, Wu C, Chen J, Wo D, Ren DN, Yan H, Peng L, Zhu W. Resveratrol prevents Ang II-induced cardiac hypertrophy by inhibition of NF-\u0026kappa;B signaling. Biomed Pharmacother. 2023 Sep;165:115275. doi: 10.1016/j.biopha.2023.115275. Epub 2023 Aug 2. PMID: 37541173.\u003c/li\u003e\n\u003cli\u003eKang DG, Sohn EJ, Kwon EK, Han JH, Oh H, Lee HS. Effects of berberine on angiotensin-converting enzyme and NO/cGMP system in vessels. Vascul Pharmacol. 2002 Dec;39(6):281-6. doi: 10.1016/s1537-1891(03)00005-3. PMID: 14567065.\u003c/li\u003e\n\u003cli\u003eLi MH, Zhang YJ, Yu YH, Yang SH, Iqbal J, Mi QY, Li B, Wang ZM, Mao WX, Xie HG, Chen SL. Berberine improves pressure overload-induced cardiac hypertrophy and dysfunction through enhanced autophagy. Eur J Pharmacol. 2014 Apr 5;728:67-76. doi: 10.1016/j.ejphar.2014.01.061. Epub 2014 Feb 6. PMID: 24508518.\u003c/li\u003e\n\u003cli\u003eRegitz-Zagrosek V, Kararigas G. Mechanistic Pathways of Sex Differences in Cardiovascular Disease. Physiol Rev. 2017 Jan;97(1):1-37. doi: 10.1152/physrev.00021.2015. PMID: 27807199.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bratislava-medical-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bratislava Medical Journal](https://link.springer.com/journal/44411)","snPcode":"44411","submissionUrl":"https://submission.springernature.com/new-submission/44411/3","title":"Bratislava Medical Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aurantiamide acetate, Hypertensive cardiac hypertrophy, MAPK signaling, Endoplasmic reticulum stress","lastPublishedDoi":"10.21203/rs.3.rs-6939373/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6939373/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eHypertensive cardiac hypertrophy contributes significantly to heart failure and is mediated by maladaptive mechanisms including mitogen-activated protein kinase (MAPK) signaling and endoplasmic reticulum (ER) stress. Aurantiamide acetate (AA), a dipeptide derivative derived from edible plants and marine fungi, exhibits anti-inflammatory and vasoprotective effects. However, its impact on hypertensive cardiac hypertrophy remains unclear. This study aims to explore the therapeutic potential of AA in mitigating cardiac hypertrophy by targeting MAPK signaling and ER stress pathways.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eCellular hypertrophy was induced in H9C2 cardiomyoblasts using angiotensin II (Ang II), and tunicamycin (TM) was used to trigger ER stress. AA\u0026rsquo;s effects were assessed via viability assays, immunofluorescence staining, and ELISA for hypertrophic and ER stress markers. In vivo, hypertensive hypertrophic cardiomyopathy (HCM) was established in rats using a two-kidney, one-clip (2K1C) model, followed by AA administration. Cardiac hypertrophy, fibrosis, MAPK activation, and ER stress markers were evaluated histologically and biochemically.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn vitro, AA significantly reduced Ang II-induced cardiomyocyte hypertrophy and ER stress, as evidenced by decreased cell size, atrial natriuretic peptide (ANP) expression, and glucose-regulated protein 78 (GRP78) levels. In vivo, AA ameliorated hypertension-induced cardiac remodelling, reduced myocardial fibrosis, suppressed MAPK phosphorylation (p38, extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK]), and alleviated ER stress. Blood pressure and serum Ang II levels were also significantly lowered in AA-treated rats.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eAurantiamide acetate mitigates hypertensive cardiac hypertrophy by inhibiting MAPK activation and ER stress both in vitro and in vivo, highlighting its potential as a novel therapeutic agent for hypertensive heart disease.\u003c/p\u003e","manuscriptTitle":"Aurantiamide Acetate Mitigates Hypertensive Cardiac Hypertrophy via Modulation of MAPK Signaling and ER Stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-04 11:15:11","doi":"10.21203/rs.3.rs-6939373/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-07T19:33:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-25T21:02:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-14T08:55:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253054233850227540265407672922400327133","date":"2025-08-12T19:22:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-03T14:11:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133381413743808227957906875062879344338","date":"2025-07-31T06:36:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320315440002769739160197910991255935692","date":"2025-07-31T03:06:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-29T20:57:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-21T08:53:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-21T08:51:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bratislava Medical Journal","date":"2025-06-20T13:23:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bratislava-medical-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bratislava Medical Journal](https://link.springer.com/journal/44411)","snPcode":"44411","submissionUrl":"https://submission.springernature.com/new-submission/44411/3","title":"Bratislava Medical Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"73d3c337-9642-474b-9eb7-36be17ca0e37","owner":[],"postedDate":"August 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T18:38:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-04 11:15:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6939373","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6939373","identity":"rs-6939373","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}