Hirsutine ameliorates obesity cardiomyopathy through the induction of mitophagy by stablizing parkin

Hirsutine ameliorates obesity cardiomyopathy through the induction of mitophagy by stablizing parkin | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 20 August 2025 V1 Latest version Share on Hirsutine ameliorates obesity cardiomyopathy through the induction of mitophagy by stablizing parkin Authors : Lu Ding , Jianshe Ma , Jingwen Wang , Ru Zhao , Ahmad Naqib shuid , Shijia Chen , Zelei Ye , … Show All … , Zichen Gao , Yi Chen , Meiyao Song , Junming Fan , Hao Zhou , and Peifeng Jin [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.175569080.03236966/v1 128 views 42 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose : Hirsutine, a potent indole alkaloid extracted from U. rhynchophylla , has been shown to ameliorate cardiac insulin resistance in high-fat diet (HFD)-induced diabetic mice. However, the underlying regulatory mechanisms remain incompletely understood. This study aims to investigate the therapeutic potential of hirsutine in obesity cardiomyopathy (OCM) and explore its associated regulatory mechanisms. Experimental Approach : We established a murine model of OCM by feeding mice a HFD for 24 weeks, followed by an 8-week treatment with hirsutine. Concurrently, palmitate-treated neonatal mouse cardiomyocytes (NMCMs) and H9c2 cells were used as the in vitro model. Cardiac function was assesed throuh invasive hemodynamic evaluations, and indices of left ventricular hypertrophy were recorded. Structural analyses were conducted using histomorphology and transmission electron microscopy (TEM). Moelcular and structural analyses were preformed using RNA sequencing, Western blotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunoprecipitation, and an activity-based protein profiling chemoproteomic approach (ABPP). Key Results : Hirsutine treatment significantly attenuated cardiac dysfunction and damage in obese mice. Mechanistic studies revealed that its therapeutic effects are mediateted through enhanced mitophagy. Inhibition of mitophagy negated hirsutine’s protective effects against obesity-related cardiomyopathy. Hirsutine treatment significantly upregulated Parkin signaling and increased mitophagy levels. The mechanism involves promoting mitophagy by regulating Parkin protein stability by binding to residues 1103-1394 of LRPPRC，acting like a molecular glue to prevent its degradation. Conclusion and Implications : These findings demonstrate that hirsutine alleviates OCM by binding to LRPPRC, thereby promoting mitophagy via the Parkin pathway. Thus, targeting LRPPRC could offer a promising strategy for hirsutine in OCM therapeutics. Hirsutine ameliorates obesity cardiomyopathy through the induction of mitophagy by stablizing parkin Lu Ding 1, 2, # , Jianshe Ma 2, # , Jingwen Wang 1, # , Ru Zhao 3 , Ahmad Naqib shuid 3 , Shijia Chen 1 , Zelei Ye 1 , Zichen Gao 1 , Yi Chen 1 , Meiyao Song 1 , Junming Fan 2 , Hao Zhou 4, *, Peifeng Jin 1, * 1 Department of Cardiac Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, Zhejiang, China. 2 School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China. 3 Advanced Medical & Dental Institute, University sains malaysia, State of Penang, Malaysia. 4 Department of Cardiology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, Zhejiang, China. # Lu Ding, Jianshe Ma, and Jingwen Wang contributed equally to this work * Corresponding authors: Hao Zhou and Peifeng Jin, The First Affiliated Hospital of Wenzhou Medical University, Nanbaixiang Street, Ouhai District, Wenzhou City, Zhejiang 325015, China. Email: [email protected] (Hao Zhou); [email protected] (Peifeng Jin). Tel:86-0577-55579242 Fax: 86-0577-55579242+661505 Running head: Hirsutine Ameliorates Cardiomyopathy by Stabilizing Parkin CONFLICT OF INTREREST The authors declare that they have no conflict of interest. ACKNOWLEDGEMENTS This work was supported by the Wenzhou Municipal Science and Technology Bureau of China (Y2022009 to PF Jin), Wenzhou Medical University Youth Talent Research Project (KYYW202311 to PF Jin), the College Students’ innovation of Science and Technology Activities Plan of Zhejiang Province (2025R413A023 to JW Wang), and the National Innovation and Entrepreneurship Training Program for College Students (202510343018 to Y Chen). DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding authorupon reasonable request. Some data may not be made available because of privacy or ethical restrictions. ABSTRACT Background and Purpose : Hirsutine, a potent indole alkaloid extracted from U. rhynchophylla , has been shown to ameliorate cardiac insulin resistance in high-fat diet (HFD)-induced diabetic mice. However, the underlying regulatory mechanisms remain incompletely understood. This study aims to investigate the therapeutic potential of hirsutine in obesity cardiomyopathy (OCM) and explore its associated regulatory mechanisms. Experimental Approach : We established a murine model of OCM by feeding mice a HFD for 24 weeks, followed by an 8-week treatment with hirsutine. Concurrently, palmitate-treated neonatal mouse cardiomyocytes (NMCMs) and H9c2 cells were used as the in vitro model. Cardiac function was assesed throuh invasive hemodynamic evaluations, and indices of left ventricular hypertrophy were recorded. Structural analyses were conducted using histomorphology and transmission electron microscopy (TEM). Moelcular and structural analyses were preformed using RNA sequencing, Western blotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunoprecipitation, and an activity-based protein profiling chemoproteomic approach (ABPP). Key Results : Hirsutine treatment significantly attenuated cardiac dysfunction and damage in obese mice. Mechanistic studies revealed that its therapeutic effects are mediateted through enhanced mitophagy. Inhibition of mitophagy negated hirsutine’s protective effects against obesity-related cardiomyopathy. Hirsutine treatment significantly upregulated Parkin signaling and increased mitophagy levels. The mechanism involves promoting mitophagy by regulating Parkin protein stability by binding to residues 1103-1394 of LRPPRC，acting like a molecular glue to prevent its degradation. Conclusion and Implications : These findings demonstrate that hirsutine alleviates OCM by binding to LRPPRC, thereby promoting mitophagy via the Parkin pathway. Thus, targeting LRPPRC could offer a promising strategy for hirsutine in OCM therapeutics. Keywords Cardiomyopathy; High fat diet; Hirsutine; Mitophagy; Parkin; LRPPRC Bullet point summary What is already known There are no satisfactory medications to treat OCM. Parkin is a crucial protein that regulates the mitophay and apoptosis. What this study adds Hirsutine is a natural product derived from Uncaria rhynchophylla, which enhanced mitophagy both in PA-induced cardiomyocytes and OCM mouse model. Hirsutine directly binds to the LRPPRC protein and stabilizes Parkin, acting like a molecular glue to prevent its degradation. What is the clinical significance Hirsutine is a novel Parkin stabilizer that offers a new therapeutic strategy for OCM. Abbreviations CT, cycle threshold;DMEM, Dulbecco’s modified Eagle’s medium;EF, ejection fractions;FBS, fetal bovine serum;FS, fractional shortening;H&E, haematoxylin & eosin;HFD, high fat diet;HW/TL, HW/tibia length;KEGG, Kyoto Encyclopedia of Genes and Genomes;LRPPRC, leucine-rich pentatricopeptide repeat-containing;LVFS, left ventricular fractional shortening;LVEF, left ventricular ejection fraction;NMCMs, neonatal mouse cardiomyocytes;OCM, obesity cardiomyopathy;PA, palmitate;qRT-PCR, quantitative reverse transcription polymerase chain reaction;TEM, transmission electron microscope;TUNEL, Terminal Deoxynucleotidyl Transferase dUTP nick-end Labeling;WGA, wheat germ agglutinin. INTRODUCTION The prevalence of obesity is rising globally, driven by lifestyle changes, dietary habits, social environments, and an aging population.(Collaborators et al., 2017). Obesity is an independent risk factor for heart failure, significantly impacting ventricular function.(Alpert, Lavie, Agrawal, Aggarwal, & Kumar, 2014; Hubert, Feinleib, McNamara, & Castelli, 1983). The American Heart Association’s 2016 Scientific Statement recognizes obesity as a potential cause of cardiac dysfunction, categorizing obesity cardiomyopathy (OCM) as a subtype of dilated cardiomyopathy with endocrine or metabolic etiology(Bozkurt et al., 2016). Approximately half of the cases of OCM are associated with heart failure and sudden cardiac death(Kaimori et al., 2024). OCM not only diminishes quality of life but also increases health risks and healthcare burdens worldwide. Current clinical management strategies for OCM primarily involve pharmacological interventions and lifestyle modifications; however, there is a notable lack of effective and targeted medications and procedures. Therefore, further exploration and identification of novel therapeutic targets and strategies are essential for medical cardiologists to effectively prevent heart failure and sudden cardiac death. Traditional Chinese medicine has demonstrated efficacy in treating metabolic cardiomyopathy(Fang et al., 2024; Huang et al., 2023; Wang et al., 2024; C. Zhang et al., 2023). One compound of particular interest is hirsutine, an indole alkaloid isolated from Uncaria rhynchophylla, a traditional Chinese herbal medicine with a long history of use in managing dyslipidemia and hypertension(J. G. Zhang et al., 2017). Contemporary research has identified hirsutine as exhibiting a broad spectrum of biological activities, including cardioprotection, glucose regulation, antihypertensive effects, antiarrhythmic properties, and anticancer activity(Bhuia et al., 2023; Jiang et al., 2023; Kang et al., 2022; Wu, Gu, Zhu, & Zhu, 2011; Zhu et al., 2015). Notably, hirsutine has shown promise in regulating glucose homeostasis, potentially mitigating hepatic and cardiac insulin resistance —a significant factor in the pathogenesis of OCM(W. Hu et al., 2022). This suggests that hirsutine could serve as a valuable candidate for the treatment of OCM. However, the specific mechanisms by which hirsutine regulates OCM remain inadequately understood. This study aims to evaluate the effects of hirsutine on OCM using both animal and cell models, while investigating the underlying molecular mechanisms. The objective is to establish a foundation for the future clinical application of hirsutine in treating patients with OCM. METHODS Animals Adult healthy C57BL/6J male mice aged 8 to 10 weeks with 23-25g body weights were obtained from the Vital River Laboratories Animal Technology Co., Ltd (Beijing, China). Mice were housed in a temperature-controlled environment (temperature: 22-23°C; humidity: 55-60%) with 12-h light/dark cycle (light on 07:00, light off 19:00) and had free access to food and water. All animal experiments were approved by the Animal Ethics Committee of Wenzhou Medical University (Approval No. 2022WY1496) and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH publication No. 8523, revised 1996). To induce OCM, mice were fed a high-fat diet (HFD; Research Diets, D12492; 60% kcal from fat) for 24 weeks, while Control mice were fed a standard chow diet (D12450J; 10% kcal from fat). After 16 weeks of HFD, mice were randomized into five groups (n = 8 per group): (1) Chow + vehicle, (2) Chow + hirsutine, (3) HFD + vehicle, (4) HFD + hirsutine, and (5) HFD + hirsutine+Midivi-1. Hirsutine (10 mg/kg/day) or vehicle (citric acid) was administered via oral gavage for an additional 8 weeks. Midivi-1(50mg/kg/, twice/week) was given via intraperitoneal injection for the same duration. At the study endpoint, mice were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and euthanized. Hearts were excised, rinsed with cold PBS and weighed. The heart weight-to-tibia length ratio (HW/TL) was calculated. Left ventricles were either snap-frozen for molecular analysis or fixed in 4% paraformaldehyde for histology. Reagents and antibodies Hirsutine (purity > 98%, #A1067) was purchased from MUST bio-technology CO.,Ltd (Chengdu, China) and dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mg/ml stock solution and was stored at -20°C. Fetal bovine serum (FBS) was obtained from ExCellBio (Shanghai, China). Protein A/G agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Information on antibodies, material and suppliers were detailed in the designated sections and listes in Supplementary Table S1. Echocardiographic analyses Transthoracic echocardiography was performed using a Vevo 2100 system (VisualSonics, Toronto, Canada) with an MS-550S transducer (22–55 MHz). Mice were anesthetized with 2% isoflurane and maintained at 37°C. M-mode images from the parasternal long axis were used to measure left ventricular (LV) end-diastolic diameter (LVEDd), end-systolic diameter (LVESd), ejection fraction (EF), and fractional shortening (FS = (LVEDd − LVESd)/LVEDd × 100%). Measurements were averaged over three cardiac cycles and analyzed by blinded observers using VevoLAB software (v5.6.1, Fujifilm VisualSonics). Histological analysis Paraffin-embedded heart tissues were sectioned (5 µm) and stained with hematoxylin and eosin (H&E), Masson’s trichrome, and wheat germ agglutinin (WGA) to evaluate cardiomyocyte hypertrophy and fibrosis. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining Apoptosis was assessed using a TUNEL assay kit (DeadEnd™ Fluorometric TUNEL System, G3250, Promega) according to the manufacturer’s instructions. Images were acquired by confocal microscopy (FluoView FV1000, Olympus, Tokyo, Japan). Transmission electron microscope (TEM) Heart tissues were cut into 1 mm³ blocks and fixed in 2.5% glutaraldehyde. Samples were processed and imaged by Service Biotech (Shanghai, China) as previously described. RNA Sequencing of the hearts The hearts were harvested from mice and the atrium and right ventricle have been removed. The sample were then incubated with RNAlater solution (NO.74704; Invitrogen). Subsequently, mRNA was extracted using FastPure Complex Tissue (RC113-01, Vazyme, Nanjing, China) and shipped to Aksomics for mRNA sequencing (n=5 biologicl replicates per group). Mouse heart mitochondrial isolation Mitochondria were isolated from mouse cardiac tissue using the Mitochondrial Isolation Kit (#SM0020, Solarbio), following the manufacturer’s instructions. Briefly, freshly excised left ventricular tissue was rinsed with ice-cold buffer and finely minced in pre-chilled isolation buffer. The homogenate was centrifuged at 1,000 × g to remove nuclei and debris, and the resulting supernatant was transferred to a fresh tube. A subsequent centrifugation at 12,000 × g was performed to pellet the mitochondria. The mitochondrial pellet was then washed with isolation buffer supplemented with a protease inhibitor cocktail (#7012, CST), followed by a final centrifugation at 12,000 × g. The purified mitochondrial fraction was collected and stored for further biochemical analyses. Cell culture and drug treatments Neonatal mouse cardiomyocytes (NMCMs) were isolated and cultured as previously described. In summary, ventricular tissue was dissected from neonatal mice, rinsed, and minced into ~1 mm³ fragments. The tissue fragments were digested using 0.2% collagenase type II (Sigma, St. Louis, MO, USA) at 37 °C for 45 minutes with gentle agitation. The digested material was filtered through a 75 μm cell strainer two to three times to ensure complete tissue dissociation. The filtrate was centrifuged at 800 × g for 5 minutes, and the pellet was resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO, Grand Island, NY, USA) supplemented with 15% fetal bovine serum (FBS). Cells were pre-plated for 1 hour at 37 °C to selectively remove adherent fibroblasts. The supernatant, enriched in cardiomyocytes, was then replated onto collagen-coated culture dishes and incubated overnight at 37 °C. After 24 hours, non-adherent cells were washed off, and the medium was refreshed. Cardiomyocytes were confirmed by sarcomeric alpha actinin immunofluorescence staining. Subsequently, cells were adjusted to a density of 1 × 10⁶ cells/mL and seeded into appropriate culture plates for further experimental procedures. The H9c2 rat cardiomyocyte cell line (Otwo Biotech Inc., RRID:CVCL_0286) and HEK 293T cells (Cat. GNHu17, RRID:CVCL_0063) was maintained in DMEM supplemented with 10% FBS under standard culture conditions. For hirsutine treatment, NMCMs were pretreated with 200 μM hirsutine for 8 hours, followed by exposure to 5 μM palmitic acid (PA) for an additional 24 hours. Unless stated otherwise, 5 μM PA was used for all in vitro experiments. Assessment of mPTP opening Mitochondrial permeability transition pore (mPTP) opening was evaluated using the calcein-AM/cobalt quenching method (Beyotime, C2009S). Cells were seeded on confocal dishes and treated as indicated. Following treatment, cells were incubated in HBSS containing 5 µM calcein-AM and 0.5 mM CoCl₂ for 15 minutes at 37 °C to quench cytosolic fluorescence. After two washes with PBS, fluorescence intensity was measured using a confocal microscope (excitation: 488 nm), with mitochondrial calcein signal serving as an indicator of mPTP status. Plasmid and cell transfection Mouse LRPPRC and PRKN cDNA were synthesized by Servicebio Co., Ltd. (Wuhan, China). Small interfering RNA (siRNA) was designed against Parkin and LRPPRC by Ribobio (Guangzhou, China). Transfections were performed according to the manufacturer’s protocol of Lipofectamine 3000 (Invitrogen, NY, USA). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from left ventricular tissues, H9c2 cells using TRIzol reagent (Invitrogen, CA, USA). cDNA synthesis was performed using the PrimeScript™ RT reagent Kit (RR037A, Takara, Kyoto, Japan) according to the manufacturer’s protocol. Quantitative PCR was carried out using SYBR Green Master Mix (Q311-02, Vazyme) on a 7900 Real-Time PCR System (Applied Biosystems, CA, USA). Gene expression of ANP, BNP, and β-MHC was normalized to GAPDH and quantified using the 2 −ΔΔCT method. All reactions were performed in duplicate. Primer sequences (GenScript Biotech Corp, Nanjing, China) were as follows: ANP: forward 5′-ACCACCTGGAGGAGAAGA-3′, reverse 5′-TTCAAGAGGGCAG ATCTATC-3′. BNP: forward 5′-GCACAAGATAGACCGGATCG-3′, reverse 5′-CCCAGGCAGA GTCAGAAAC-3′. β-MHC: forward 5′-TTTGATGTGCTGGGCTTCAC-3′, reverse 5′-TGACATACTCG TTGCCCACT-3′. GAPDH: forward 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse 5′-TGTAGACCA TGTAGTTGAGGTCA-3′. Western blotting and immunoprecipitation assays Proteins were extracted from tissues or cultured cells using RIPA buffer (Beyotime Biotechnology, Shanghai) supplemented with protease and phosphatase inhibitors (Roche, Switzerland). Lysates were centrifuged at 12,000 × g for 15 minutes at 4 °C, and protein concentration was quantified using the BCA assay (Thermo Scientific, MA, USA). Equal amounts (30 μg) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, MA, USA). Membranes were blocked in 5% non-fat milk for 1 hour and incubated overnight with primary antibodies at 4 °C. After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour and visualized using ECL detection reagent (Thermo Scientific, MA, USA). Images were captured using a Bio-Rad imaging system and analyzed with ImageJ software. GAPDH was used as the internal control. For immunoprecipitation, lysates were incubated with 1 μg of specific antibody or control IgG overnight at 4 °C. Protein A/G agarose beads were added for 4 hours, followed by three washes with cold IP buffer. The bound protein complexes were eluted with loading buffer and analyzed by Western blot. Activity-based protein profiling synthesis of hirsutine‑conjugated magnetic microbeads To prepare hirsutine-conjugated magnetic microbeads, 10 mg of amino-functionalized beads (Yike Biological Co., Shanghai, China) were added to a reaction tube along with BOC₂O (1.02 mmol), hirsutine (1.02 mmol), DMAP (0.04 mmol), and 2,6-dimethylpyridine in acetonitrile. Carboxyl magnetic beads were added to reach a final volume of 2 mL. The mixture was stirred at 28 °C under nitrogen for 24 hours. The crude product was washed with 0.5 M HCl and saturated NaHCO₃, dried over MgSO₄, and purified by short silica gel chromatography using CH₂Cl₂ as the eluent. Beads were blocked in 0.3% casein in PBST (pH 7.4) and stored in 0.25% BSA in PBS (pH 7.4). Mass spectrometry analysis of drug-protein interactions To characterize the conjugation of hirsutine to magnetic beads and identify potential protein-binding partners, magnetic bead–drug complexes were synthesized via an amide bond formation reaction. Briefly, BOC₂O (1.02 mmol) and the drug molecule hirsutine (1.02 mmol) were dissolved in acetonitrile (MeCN) in the presence of the catalyst DMAP (0.04 mmol) and 2,6-dimethylpyridine. Carboxylated magnetic beads (10 mg) were then added to the solution, and the mixture was stirred at 28 °C under a nitrogen atmosphere for 24 hours. The reaction mixture was concentrated under reduced pressure, and the product was washed sequentially with 0.5 M HCl and saturated NaHCO₃. The beads were dried over MgSO₄ and purified using short-column silica gel chromatography with dichloromethane (CH₂Cl₂) as the elution solvent, yielding a crystalline product. The surface charge of the beads was assessed using a nanoparticle zeta potential analyzer. A shift from –37.1 mV (unmodified beads) to –25.9 mV (hirsutine-conjugated beads) indicated successful drug attachment via interaction between carboxyl groups on the beads and functional groups on hirsutine. For target identification, both hirsutine-conjugated and control (unmodified) beads were incubated overnight at 4 °C with cellular protein lysates. After incubation, beads were washed thoroughly to remove non-specifically bound proteins. Bound proteins were eluted, resolved by SDS-PAGE, and visualized by Coomassie brilliant blue staining. Distinct protein bands were excised and analyzed by mass spectrometry (Yike Biological Co., Shanghai, China) to identify potential hirsutine-interacting proteins. Surface plasmon resonance (SPR) The interaction between HS and Lrpprc (cat#ep013105hu1; CUSA Biological, Wuhan, China) was determined using a Biacore T200 instrument (Cytiva; Marlborough, MA, USA) with a CM5 sensor chip (Cytiva). Briefly, Lrpprc was loaded onto the sensors using an amine-coupling kit (Cytiva). HS samples (at concentrations of 0, 0.391, 0.781, 1.563, 3.125,307 or 6.250 μM) were prepared in running buffer (PBS, 0.5% P20, 5% DMSO). The sensor and sample plates were placed on the instrument and the ELE samples flowed over the black and target sensors. Six concentrations were injected successively, at a flow rate of 30 μL·min-1,for a 200 s association phase, which was followed by a 100 s dissociation phase at 25 ℃. The final graphs were obtained by subtracting the blank sensorgrams and blank samples from the duplex. The data were analyzed using Biacore T200 software EV (Cytiva). The dissociation constant (KD) was calculated by global fitting of kinetic data from various concentrations of HS using a 1:1 binding model. Immunofluorescence staining Immunofluorescence analysis was performed on paraffin-embedded heart tissue sections as well as H9c2 and NMCMs cultured on glass coverslips. For mitochondrial visualization, live H9c2 cells were incubated with MitoTracker Red (500 nM) at 37 °C for 30 minutes, followed by fixation in 4% paraformaldehyde (PFA). Cell membranes were permeabilized with 0.1% Triton X-100 for 5 minutes at room temperature. After blocking, cells were incubated overnight at 4 °C with LC3B and phalloidin primary antibodies (1:100 dilution). The next day, cells were washed and incubated with appropriate fluorescent secondary antibodies for 1 hour at room temperature. Nuclei were counterstained where applicable. Fluorescent signals were captured using a confocal laser scanning microscope. Molecular docking analysis The full-length structure of Lrpprc was retrieved from the AlphaFold Protein Structure Database (ID: P42704), and the structure of Hirsutine was downloaded from PubChem.Protein ligands and water molecules were removed using PyMOL. Hydrogen atoms were added, amino acids were optimized, and the active site was modeled with AutoDock Tools1.5.7. Molecular docking was performed using AutoDock 4.2.6 with default parameters for all settings. The docking results were visualized with PyMOL Statistical analysis All data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA). Comparisons between two groups were conducted using an unpaired Student’s t -test. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was applied. A P value less than 0.05 was considered statistically significant. 3. Results 3.1 Hirsutine attenuates cardiac disfunction and cardiac damage in obesity mice To explore whether hirsutine could protect the heart from damage caused by a long-term high-fat diet (HFD), we treated mice with hirsutine after 16 weeks on an HFD and continued the treatment for 8 weeks( Fig. 1A ). HFD feeding led to a moderate increasing in body weight (BW) compare to the chow-fed controls, HS treatment did not affect the BW (Figure S1). As expected, mice on the HFD developed signs of cardiac hypertrophy. Their heart weight relative to tibia length (HW/TL) was significantly higher compared to normal controls. However, treatment with hirsutine notably reduced this ratio ( Fig. 1B ), suggesting a protective effect against heart enlargement. Microscopic analysis supported these findings. Mice on the HFD had visibly enlarged heart cells, but this was significantly reduced in those that received hirsutine. In addition, heart tissue from HFD-fed mice showed increased fibrosis—a marker of chronic heart stress—which was also noticeably less severe following hirsutine treatment. Beyond reducing structural damage, hirsutine helped improve heart function. Mice on the HFD showed impaired heart performance, as measured by echocardiography. Specifically, both left ventricular fractional shortening (LVFS) and ejection fraction (LVEF) were reduced—hallmarks of cardiac dysfunction. Hirsutine treatment significantly improved both measures ( Fig. 1C–G ), suggesting that it not only prevents damage but also helps restore proper heart function. We examined two molecular markers of heart hypertrophy, ANP and BNP, both of which were elevated in HFD-fed mice, indicating heart strain. Notably, hirsutine reduced their expression levels toward normal (Fig. 1H, I), highlighting its therapeutic potential. In summary, hirsutine was effective in reducing HFD-induced heart enlargement and fibrosis, lowering stress-related heart markers, and improving overall cardiac performance in mice. 3.2 Hirsutine improves mitochondrial bioenergetics and reduces apoptosis in the hearts of HFD mice To better understand how hirsutine protects the heart HFD-induced damage, we conducted transcriptome sequencing of cardiac tissues from HFD-fed mice, with and without hirsutine treatment. Compared to the HFD group, the HFD + hirsutine group showed significant changes in gene expression, with 245 genes downregulated and 71 genes upregulated (Fold change > 1.2, P < 0.05; Fig. 2A ). Gene Ontology enrichment analysis indicated that the differentially expressed genes primarily influenced mitochondrial function( Fig.2B ).Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that hirsutine treatment was associated with mitophagy( Fig.2C ). Additionally, gene set enrichment analysis (GSEA) confirmed significant alterations in key mitochondrial processes, particularly apoptosis, which were more active in the HFD + hirsutine group compared to the HFD-only group (Fig. 2D). This suggests that hirsutine improves mitochondrial function and reduces cell death. To directly examine mitochondrial health, we performed transmission electron microscopy (TEM) on cardiac tissues. In the hearts of HFD-fed mice, we observed extensive mitochondrial damage, including swelling, membrane disruption, and loss of cristae. In contrast, hirsutine-treated mice exhibited significantly fewer damaged mitochondria. Furthermore, there was a marked increase in autophagosomes and autolysosomes containing mitochondrial debris in the HFD + hirsutine group, indicating enhanced mitophagy and cellular cleanup processes ( Fig. 2E ). Western blotting of isolated mitochondrial fractions supported these findings: mitophagy-related proteins were suppressed in the HFD group but restored with hirsutine treatment( Fig. 2F, G ). Conversely, apoptosis-related mitochondrial proteins were elevated in HFD mice and significantly decreased following hirsutine administration ( Fig. 2H, I ). TUNEL staining of heart tissues confirmed increased cardiomyocyte apoptosis in HFD-fed mice, which was markedly reduced in those treated with hirsutine (Fig. 2J). This reduction in cell death aligns with the protein expression data. In summary, these results suggest that hirsutine mitigates HFD-induced cardiac dysfunction by preserving mitochondrial structure, enhancing metabolic function, promoting mitophagy, and reducing apoptosis in heart tissue. 3.3 Hirsutine attenuates PA-induced mitophagy and apoptosis in H9c2 cardiomyocytes To investigate the role of mitophagy in the hirsutine-mediated response to PA-induced cardiomyocyte stress, we examined the autophagy biomarker LC3 in neonatal mouse cardiomyocytes (NMCM) and H9c2 cells. Confocal microscopy revealed that hirsutine treatment significantly increased LC3 fluorescence intensity( Fig. 3A ). The co-localization of LC3 (green) with Mitotracker (red) was markedly enhanced in H9c2 cells treated with hirsutine compared to those treated with PA alone ( Fig. 3B ). We assessed mitochondrial permeability transition pore (mPTP) activation using Calcein-AM staining. Following PA incubation, mPTP activition was more pronounced in the PA group than in the hirsutine group ( Fig. 3C, D ). Additionally, TUNEL staining indicated that PA increased cardiomyocyte apoptosis, which was significantly reduced following hirsutine treatment (Fig. 3 E, F). Together, these findings suggest that hirsutine not only prevents structural hypertrophy but also mitigates cellular and molecular markers of cardiomyocyte stress under lipotoxic conditions. 3.4 Inhibition of mitophagy reverses the therapeutic roles of hirsutine in HFD-induced cardiomyopathy Building on our previous findings that hirsutine enhances mitophagy, we investigated whether inhibiting mitophagy would negate hirsutine’s benefits in HFD-induced cardiomyopathy. Midivi-1, a mitophagy inhibitor, was administered alongside hirsutine (Fig. 4A). Our results showed that mitophagy inhibition blocked hirsutine’s protective effects, as indicated by increased heart weight to tibia length (HW/TL) ratios and larger cardiomyocyte cross-sectional areas compared to the hirsutine+HFD group. Cardiac fibrosis was also significantly elevated in Midivi-1-treated mice. Functionally, Midivi-1 treatment led to worsened cardiac dilation and dysfunction, with marked reductions in fractional shortening and ejection fraction (Fig. 4B-G). Furthermore, Midivi-1 increased the expression of ANP and BNP in HFD-fed mice (Fig. 4H,I). These findings confirm that inhibiting mitophagy negates the cardioprotective effects of hirsutine in the context of HFD-induced cardiomyopathy. 3.5 Hirsutine effects on mitophagy of cardiomyocyte via PINK1/Parkin pathway. To investigated the potential mechanisms underlyling hirsutine’s effect on mitophagy,we performed ABPP to identify potential downstream effectors. KEGG analysis using the Metascape database indicated that hirsutine may interact with several proteins associated with the Parkin ubiquitin-proteasomal system pathway (Fig. 5A-B). Parkin, an E3 ubiquitin ligase, is recruited to damaged mitochondria by PINK1, and the PINK1/Parkin pathway is crucial for mitochondrial quality control. We assessed the effect of hirsutine on PINK1/Parkin signaling. Western blotting demonstrated that PINK1 and Parkin levels were increased in hirsutine-treated mice and H9c2 cells (Fig. 5C-F and Fig.S2). To further validate Parkin’s role in cardiomyocyte hypertrophy, we conducted in vitro experiments using H9c2 cells transfected with siRNA targeting Parkin (siParkin). Mitophagy was mitigated by siParkin (FigS3). We found that mRNA levels of hypertrophic markers ANP and BNP were significantly elevated in the PA + siParkin group compared to PA alone. Conversely, hirsutine treatment reduced the PA-induced upregulation of these markers, but this protective effect was reversed by siParkin treatment (Fig. 5G). Consistently, siParkin exacerbated PA-induced cardiomyocyte hypertrophy, as shown by increased cell size compared to PA-treated cells. Hirsutine effectively attenuated this hypertrophy, but its effect was again reversed when Parkin was knocked down (Fig. 5H, I). Together, these results demonstrate that Parkin acts as a critical positive regulator in HFD-induced cardiomyopathy and fibrosis. Furthermore, the cardioprotective effects of hirsutine appear to be partly mediated through the activation of the Parkin pathway. 3.6 Hirsutine directly binds to the LRPPRC protein. Since we found that hirsutine’s therapeutic effects in cardiomyopathy and cardiomyocyte hypertrophy involve activation of the Parkin pathway, we next sought to uncover the upstream mechanisms regulating Parkin. To do this, we performed immunoprecipitation to pull down all proteins bound to hirsutine in H9c2 cells, followed by mass spectrometry (MS) analysis to identify potential hirsutine targets Next, using the BioGRID database, we gathered differentially expressed proteins from the affinity selection mass spectrometry of Parkin. As shown by Venn diagram (Fig. 6A), among the candidates, LRPPRC, a key regulator of mitochondrial gene expression linked to mitochondrial diseases, showed strong binding affinity to hirsutine, confirming it as a direct hirsutine target. To further investigate the interaction between hirsutine and LRPPRC, we performed molecular docking simulations (Fig. 6B). The results revealed that hirsutine fits into the active pocket of LRPPRC with a binding energy of -8.022 kcal/mol. Key amino acids involved in the binding include ILE-1211, PRO-1213, PHE-1216, ASN-1208, GLU-1207, and LYS-1209 (Fig. 6C).To confirm the binding of hirsutine to LRPPRC, we performed a magnetic bead pull-down assay in H9c2 cells. As shown in Fig. 6D, whole cell lysate and hirsutine‑conjugated beads with strong band, a weak LRPPRC band in the pro-conjugated magnetic beads (Pro Beads) group was observed, while no band was seen in the empty magnetic beads (Empty Bead, negative control). To further validate LRPPRC’s role in cardiomyocyte hypertrophy, we performed in vitro experiments using H9c2 cells transfected with siRNA targeting LRPPRC (si LRPPRC). We found that mRNA levels of hypertrophic markers ANP and BNP were significantly increased in the PA+siLRPPRC group compared to PA alone. Conversely, hirsutine treatment reduced the PA-induced upregulation of these markers; however, this protective effect was reversed by si LRPPRC treatment (Fig. 6E). Consistently, siLRPPRC exacerbated PA-induced cardiomyocyte hypertrophy, as shown by increased cell size compared to PA-treated cells. Hirsutine effectively attenuated this hypertrophy, but its effect was again reversed when LRPPRC was knocked down (Fig. 6F, G). Overall, these findings highlight the crucial role of LRPPRC in mediating the protective effects of hirsutine against cardiomyocyte hypertrophy. 3.7 Hirsutine binds to the residues 1103-1394 of LRPPRC to maintain the parkin stability Based on the above experimental results, we explored the role of hirsutine in the LRPPRC-Parkin interaction in cardiomyocytes under stress. As an E3 ubiquitin ligase, the stability of Parkin is crucial for the effective regulation of mitophagy and the maintenance of cellular health. We then assessed whether hirsutine could affected the stability of Parkin. After transfecting HEK293T cells with Flag-LRPPRC and GFP-Parkin plasmids, as predicated, hirsutine increased the half-life of Parkin from approximatly 4 hrs to over 6 hrs (Fig. 6H, I). To identify which domain of LRPPRC interacts with hirsutine, we generated three truncated LRPPRC fragments based on UniProt data: LRPPRC1-653 (M1), LRPPRC654-1102 (M2), and LRPPRC1103-1394 (M3) (Fig. 6J). HEK293T cells were transfected with full-length (FL) or these truncated constructs, followed by Co-IP using hirsutine-conjugated magnetic microbeads. The results showed that hirsutine binds to the M3 fragment and full-length LRPPRC, but not to M1 or M2 fragments (Fig. 6K). This suggests that the amino-terminal region spanning residues 1103-1394 mediates the interaction with Parkin. Notably, we found that deletion residues 1103-1394 in LRPPRC limited the the effect of hirsutine on stability of Parkin protein (Fig. 6L, M). In summary, these results indicate that hirsutine regulates the stability of Parkin protein through binging the residues 1103-1394 of LRPPRC. DISCUSSION This study elucidates the critical role of hirsutine in the development of OCM. The major findings are as follows:1) Hirsutine was shown to attenuate cardiac dysfunction and damage in obese mice. 2) Mechanistic studies revealed its therapeutic effects through the enhancement of mitophagy. 3) Inhibition of mitophagy in these mice negated the protective effects of hirsutine against obesity-related cardiomyopathy. 4) Hirsutine regulates the stability of Parkin protein by binging the residues 1103-1394 of LRPPRC. (Figure 7). Cardiomyopathy remains a significant global health burden, ranking among the leading causes of heart failure and mortality worldwide. This complex disease is characterized by structural and functional abnormalities of the myocardium that compromise cardiac output and lead to life-threatening complications. The underlying causes of cardiomyopathy are multifactorial and include metabolic disturbances, infections, endocrine dysfunction, mitochondrial abnormalities, and genetic predispositions. Despite considerable advances in understanding these pathogenic mechanisms, there remains a lack of effective targeted therapies to halt or reverse the progression of cardiomyopathy. Therefore, the discovery of novel therapeutic targets and agents is of critical importance to improve clinical outcomes. In recent years, traditional Chinese medicines have shown unique advantages and enormous potential in the prevention and treatment of cardiovascular diseases due to its multi-targets and effective regulatory effects, providing new choices for the treatment of obese cardiomyopathy patients. Hirsutine belongs to traditional Chinese medicine and exhibits multifunctional properties in the treatment of various diseases. The therapeutic potential of hirsutine has been extensively investigated in preclinical studies, demonstrating its efficacy in treating cardiovascular diseases (Jiang et al., 2023; Wu et al., 2011; Xie et al., 2022), though its regulatory mechanisms are needed further clarified. Therefore, we attempted to investigate the role and potential mechanism of hirsutine in the treatment of cardiomyopathy. As expected, hirsutine was found to exert therapeutic effects against cardiomyopathy induced by HFD, as supported by a significant reduction in HW/BW ratio, a substantial decrease in cell cross-sectional area and fibrosis area, and a profound downregulation of ANP and BNP expression in the cardiac tissue, while an obvious increase in LVEF and LVFS values. Obesity often leads to impaired mitochondrial function, resulting in reduced ATP production and increased oxidative stress. Mitochondrial dysfunction in obesity is associated with a higher risk of heart failure due to decreased energy supply to cardiac muscle(Asakura et al., 2025; M. Q. Hu et al., 2025; Li et al., 2025; Ramachandra et al., 2021). Mitophagy helps maintain cellular health by removing dysfunctional mitochondria. It is essential for energy metabolism and enables cells to adapt to stress, thereby preventing apoptosis. Multiple studies have indicated that regulating mitophagy can serve as a therapeutic target for OCM(Rabinovich-Nikitin, Cogan, & Kirshenbaum, 2021; Tong et al., 2023; Tong et al., 2021).Intriguingly, the therapeutic effects of hirsutine against cardiomyopathy are linked to the promotion of mitophagy and the prevention of apoptosis in both mouse hearts and in vitro cell models. We identified that hirsutine could promoting mitophagy through the Pink/Parkin pathway by mass spectrometry scaning. Parkin knockdown abolished the protective effect of hirsutine against cardiac hypertrophy. While the precise molecular mechanism underlying hirsutine’s promotion of mitophagy remains unclear. Among the candidate proteins that interact with hirsutine and Parkin, LRPPRC stands out as a multifunctional protein predominantly localized in mitochondria. Increasing studies have shown that LRPPRC mutation or deficiency is involved in the progression of various diseases such as tumors(Xue et al., 2025; Yu et al., 2024; Zhou et al., 2023), neurodegenerative diseases(J. Zhang, Liu, Wu, Wei, & Qu, 2025), and mitochondrial diseases(Rubalcava-Gracia et al., 2024; Tang et al., 2025). Notably, LRPPRC knockdown abrogated the inhibitory effect of hirsutine on mRNA levels of ANP and BNP as well as cardiomyocyte hypertrophy induced by PA. As an E3 ubiquitin ligase, the stability of Parkin is crucial for the effective regulation of mitophagy and the maintenance of cellular health. Some studies suggest that reactive oxygen species (ROS) can induce oxidative modifications to Parkin, potentially altering its conformation and activity. These modifications may impair Parkin’s ability to ubiquitinate target proteins, subsequently leading to a reduction in mitophagy. Leyuan Liu demonstrated that LRPPRC can maintain the stability of Parkin. In our study, we found that hirsutine enhances this stabilizing function acting like a molecular glue to prevent its degradation.Molecularly, the amino-terminal region comprising amino acids 1103-1394 of LRPPRC functions as a peptide that interacts with hirsutine. When the amino acid fragments 1103-1394 of LRPPRC were truncated, the stabilizing function of hirsutine on Parkin was attenuated. Despite several novel findings presented here, there are still some limitations existed in the present study. First, we only studied the therapeutic effect of hirsutine on HFD-induced cardiomyopathy model, and the role of hirsutine in other cardiomyopathy models was not yet addressed. Second, we only focused primarily on studying cardiomyocytes and did not specifically investigate the effects of hirsutine on other cells in cardiac tissue, such as fibroblasts and macrophages. Obviously, future investigations are needed to explore and compare the ameliorative effects of hirsutine in other cardiomyopathy models and different cell types in cardiac tissue. CONCLUSION In summary, our findings demonstrate for the first time that hirsutine may serve as a mitophagy protector to therapy HFD-induced cardiomyopathy, providing a potential therapeutic approach for obesity cardiomyopathy treatment. Additionally, LRPPRC was found to be a target for hirsutine, which can directly interact with Parkin signaling pathway and play a role in regulating mitophagy in cardiomyocytes. The present results extend our understanding of the pathogenesis of HFD-induced cardiomyopathy, and LRPPRC-Parkin axis represents a promising therapeutic target for hirsutine in the treatment of HFD-induced cardiomyopathy. Ethics approval and consent to participate All animal experiments were approved by the Animal Ethics Committee of Wenzhou Medical University (Approval No. 2022WY1496) and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH publication No. 8523, revised 1996). Consent for publication This manuscript has not been published or presented elsewhere in part or in entirety, and is not under consideration by another journal. All the authors have approved the manuscript and agree with submission to your esteemed journal. AUTHOR CONTRIBUTIONS PF Jin and H Zhou : Secured funding, directed the project and reviewed the experimental results. L Ding and JM Fan: Analyzed the data, made statistical charts, wrote and edited the manuscript. L Ding , JS Ma , JW Wang, Ru Zhao, Ahmad Naqib shuid, ZL Ye, SJ Chen, ZC Gao, Y Chen, MY Song : Performed the animal model, cell culture, sample collection and experiments research. All authors contributed to and have approved the final manuscript. DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design& Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers and other organizations engaged with supporting research. References Alpert, M. A., Lavie, C. J., Agrawal, H., Aggarwal, K. B., & Kumar, S. A. (2014). Obesity and heart failure: epidemiology, pathophysiology, clinical manifestations, and management. 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(A) The experimental flowchart for the high-fat diet induced OCM mouse model. (B) The ratio of left ventricle weight to tibia length (mg/mm). (C) Representative echocardiographic images of hearts from different groups. (D) Representative images of heart (E) Representative images of heart sections stained with wheat germ agglutinin (WGA), Scale bars,50μm. (F) . Representative images of Masson’s trichrome staining of the left ventricle from mice. Scale bars,100μm. (G) Quantitative analysis of the cross-sectional area based on WGA staining in (E). (H) Quantification of the percentage of fibrosis area based on Masson’s trichrome staining in (F). (I) Quantification of left ventricle fractional shortening. (J) Quantification of left ventricle ejection fraction. (K) Representative immunoblots of ANP and BNP protein expression in cardiac tissues tested by western blotting. (L) Quantification of ANP and BNP expression levels in (K), and GAPDH was used as loading control. Data are presented as the mean ± SEM; n=4–8 mice per group. * P <0.05, ** P <0.01, *** P <0.001. Fig. 2. Hirsutine improves mitochondrial bioenergetics and reduces apoptosis in the hearts of HFD mice. (A) Volcano plots show the distribution of DEGs in the hearts between HFD and HFD+HS groups. (B, C) The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and GO terms analysis of DEGs in the hearts between HFD and HFD+HS groups. (D) Enrichment plot for the hallmark GSEA dysregulated in the hearts between HFD and HFD+HS groups. Significance threshold set at FDR<0.05. (E) Representative TEM images of heart sections of mice in different groups. Arrows indicate autophagosomes or autolysosomes containing the mitochondrial fraction. Stars indicate damaged mitochondria. Scale bars,1μm. (F, G) Representative immunoblots and quantification of autophagy-associated protein expression of mitochondrial fractions in cardiac tissues tested by western blotting. (H) Representative images of TUNEL assay in the heart sections of mice in different groups. Scale bars,50μm. (I, J) Representative immunoblots and quantification of Cyto C and C-Caspase 3 protein expression of mitochondrial fractions in cardiac tissues tested by western blotting. Data are presented as the mean ± SEM; n=4–8 mice per group. * P <0.05. Fig. 3. Hirsutine attenuates PA-induced H9c2 cells mitophagy and apoptosis. (A) Representative images of immunofluorescence staining of NMCMs (Green, α-sarcomeric actin; Red, LC3). Scale bars, 25μm. (B) Representative images of immunofluorescence staining of NMCMs (Green, LC3; Red, mitotracker). Scale bars, 100μm. (C, D) Representative images and quantitative analysis of mPTP activation in H9c2 cells assessed by Calcien-AM staining. Scale bars, 50μm. (E, F) Representative images and quantitative analysis of TUNEL assay in H9c2 cells. Scale bars, 300μm. Data are presented as the mean ± SEM; n=4–8 mice per group. * P <0.05, ** P <0.01. Fig. 4. Inhibition of mitophagy reverses the therapeutic roles of hirsutine in HFD-induced cardiomyopathy. (A) The experimental flowchart for the high-fat diet induced OCM mouse model. (B) The ratio of left ventricle weight to tibia length (mg/mm). (C) Representative echocardiographic images of hearts from different groups. (D) Representative images of heart. (E) Representative images of heart sections stained with wheat germ agglutinin (WGA), Scale bars,50μm. (F) . Representative images of Masson’s trichrome staining of the left ventricle from mice. Scale bars,100μm. (G) Quantitative analysis of the cross-sectional area based on WGA staining in (E). (H) Quantification of the percentage of fibrosis area based on Masson’s trichrome staining in (F). (I) Quantification of left ventricle fractional shortening. (J) Quantification of left ventricle ejection fraction. (K) Representative immunoblots of ANP and BNP protein expression in cardiac tissues tested by western blotting. (L) Quantification of ANP and BNP expression levels in (K), and GAPDH was used as loading control. Data are presented as the mean ± SEM; n=4–8 mice per group. * P <0.05, ** P <0.01, *** P <0.001. Fig. 5. Hirsutine effects on mitophagy of cardiomyocyte via PINK1/Parkin pathway. (A) Schematic diagram of the experimental procedure used to identify potential targets of hirsutine in H9c2 cells. (B) Kyoto Encyclopedia of Genes and Genomes enrichment analyses. (C, D ) Representative immunoblots and quantification of Pink1 and Parkin in cardiac tissues tested by western blotting. (E, F ) Representative immunoblots and quantification of Pink1 and Parkin in H9C2 cells tested by western blotting. (G) Quantification of ANP mRNA and BNP mRNA expression levels in cultured H9c2 cells tested by qRT-PCR. (H )The representative images of immunofluorescence staining of phalloidin in cultured H9c2 cells. (I) Quantitative analysis of the surface area of cardiomyocytes in (H). Data are presented as the mean ± SEM; n=5 per group. * P <0.05, ** P <0.01, *** P <0.001. Fig. 6. Hirsutine directly binds to the LRPPRC protein. (A) Venn diagram illustrating the identification of LRPPRC through mass spectrometry analysis. (B) Predicted 3D structure of hirsutine binding to LRPPRC. (C) 2D interaction illustration of hirsutin and LRPPRC. (D) Surface plasmon resonance (SPR) analysis between HS and LRPPRC.Representative immunoblot of LRPPRC protein expression in cultured H9c2 cells tested by western blotting. (E) Quantification of mRNA levels of ANP, BNP, and ꞵ-MHC expression in cultured H9c2 cells tested by qRT-PCR. (F )The representative images of immunofluorescence staining of phalloidin in cultured H9c2 cells. Scale bars, 100μm. (G) Quantitative analysis of the surface area of cardiomyocytes in (I). Data are presented as the mean ± SEM; n=3 per group. * P <0.05, ** P <0.01, *** P <0.001. Fig. 7. Hirsutine regulates of the stability of Parkin via its interaction with LRPPRC (A) Parkin levels in HEK293T cells transfected with LRPPRCfollowed by treated with CHX for different time points with DMSO or hirsutin treatment. (B) Schematic diagram of the full length (FL) and truncated versions of LRPPRC. (C) HEK293T cells were transfected with LRPPRC FL and plasmids encoding LRPPRC 1-653 , LRPPRC 654-1102 and LRPPRC 1103-1394 , respectively, and cellular lysates were immunoprecipitated with anti-flag antibody. (D) Representative western blots with Flag after Co-IP of full length and truncated forms of LRPPRC from HEK293T whole-cell lysates using Flag antibody. (E,F) Parkin levels in HEK293T cells transfected with LRPPRC Δ1103-1394 followed by treated with CHX for different time points with DMSO or hirsutin treatment. Data are presented as the mean ± SEM; n=3 independent repeats. * P <0.05. Figure 8. Graphical Abstract. The proposed mechanism of hisutine-mediated induction of the mitophagy via LRPPRC/Parkin pathway in OCM. Information & Authors Information Version history V1 Version 1 20 August 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Lu Ding The First Affiliated Hospital of Wenzhou Medical University View all articles by this author Jianshe Ma Wenzhou Medical University School of Basic Medical Sciences View all articles by this author Jingwen Wang The First Affiliated Hospital of Wenzhou Medical University View all articles by this author Ru Zhao University sains malaysia View all articles by this author Ahmad Naqib shuid University sains malaysia View all articles by this author Shijia Chen The First Affiliated Hospital of Wenzhou Medical University View all articles by this author Zelei Ye The First Affiliated Hospital of Wenzhou Medical University View all articles by this author Zichen Gao The First Affiliated Hospital of Wenzhou Medical University View all articles by this author Yi Chen The First Affiliated Hospital of Wenzhou Medical University View all articles by this author Meiyao Song The First Affiliated Hospital of Wenzhou Medical University View all articles by this author Junming Fan Wenzhou Medical University School of Basic Medical Sciences View all articles by this author Hao Zhou The First Affiliated Hospital of Wenzhou Medical University View all articles by this author Peifeng Jin [email protected] The First Affiliated Hospital of Wenzhou Medical University View all articles by this author Metrics & Citations Metrics Article Usage 128 views 42 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Lu Ding, Jianshe Ma, Jingwen Wang, et al. 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