A Multifunctional Therapeutic Peptide Attenuates Post-Myocardial Infarction Remodeling Through Antioxidant, Pro-Angiogenic, and Immunomodulatory Mechanisms

A Multifunctional Therapeutic Peptide Attenuates Post-Myocardial Infarction Remodeling Through Antioxidant, Pro-Angiogenic, and Immunomodulatory Mechanisms | 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 A Multifunctional Therapeutic Peptide Attenuates Post-Myocardial Infarction Remodeling Through Antioxidant, Pro-Angiogenic, and Immunomodulatory Mechanisms Hsin-Ying Lu, Hsiu-Yi Chu, Chao-Wei Chao, Chun-Yang Cheng, Chih-Hung Huang, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8334699/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Myocardial infarction (MI) triggers excessive oxidative stress, inflammatory activation, and maladaptive fibrosis, leading to adverse ventricular remodeling and heart failure. Therapeutic strategies capable of simultaneously modulating oxidative, inflammatory, and angiogenic pathways remain limited. This study investigates the cardioprotective effects of a novel Multifunctional Peptide (MFP) engineered to possess antioxidant, anti-inflammatory, pro-angiogenic, and endothelial-regenerative activities in a rat model of MI. Methods: Adult male Sprague–Dawley rats underwent left anterior descending (LAD) coronary artery ligation to induce MI, followed by intramyocardial administration of MFP into peri-infarct myocardium. Cardiac remodeling and function were assessed via echocardiography and histopathology at 4 weeks post-MI. Macrophage phenotypes, fibrosis, angiogenesis, and apoptosis were evaluated using immunohistochemistry. In vitro studies examined MFP-mediated cytoprotection in H9C2 cardiomyocytes under oxidative stress and its effects on macrophage polarization in RAW 264.7 cells. RNA-sequencing was performed to identify transcriptomic signatures regulated by MFP. Results: MFP treatment significantly improved left ventricular systolic function and attenuated post-MI structural deterioration. Histological analyses showed reduced cardiomyocyte apoptosis, diminished interstitial fibrosis, and markedly increased capillary density. MFP decreased pro-inflammatory macrophage infiltration while promoting reparative M2 macrophage polarization in vivo. In vitro, MFP protected cardiomyocytes against oxidative damage and enhanced M2-polarizing signaling in macrophages. Transcriptomic profiling revealed downregulation of genes associated with necrosis, inflammation, and adverse remodeling, and upregulation of pathways related to tissue repair, angiogenesis, and immune modulation. Conclusion: MFP confers robust cardioprotection after MI by coordinating the suppression of oxidative stress and inflammation with the enhancement of angiogenic and reparative pathways. These findings support MFP as a promising therapeutic candidate for limiting infarct injury and improving myocardial repair. Multifunctional peptide myocardial infarction oxidative stress macrophage polarization angiogenesis cardiac remodeling immunomodulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide, primarily resulting from the abrupt occlusion of coronary arteries due to atherosclerotic plaque rupture and subsequent thrombus formation[ 1 – 4 ]. This ischemic event leads to an immediate and profound reduction in oxygen and nutrient supply to the myocardium, triggering a cascade of pathological processes that extend far beyond the initial vascular insult[ 3 , 5 – 7 ]. The pathological progression following myocardial injury encompasses myocardial ischemia, cardiomyocyte apoptosis, fibrosis, and exacerbation of the inflammatory response[ 8 ], collectively contributing to ventricular remodeling and structural and functional abnormalities and loss of functional cardiomyocytes within the heart[ 9 , 10 ], leading to more than 50% five-year mortality rate after MI[ 11 – 13 ]. At the cellular level, ischemia instigates mitochondrial dysfunction and excessive production of reactive oxygen species (ROS), creating a highly oxidative environment that exacerbates cellular injury[ 14 , 15 ]. Oxidative stress, in turn, disrupts calcium homeostasis, damages cellular membranes, and activates intrinsic apoptotic pathways, leading to widespread cardiomyocyte death through both apoptosis and necrosis[ 16 ]. The resulting loss of functional myocardium impairs contractile performance and initiates the remodeling of the left ventricle. This initial injury is further complicated by a robust inflammatory response[ 8 ], characterized by the recruitment and activation of neutrophils, macrophages, and other immune cells[ 17 ]. While inflammation is essential for debris clearance and initiation of repair, excessive or prolonged inflammatory activity contributes to further tissue damage, promotes fibrotic scar formation, and impairs regenerative potential[ 17 ]. The transition from inflammation to resolution and repair is often dysregulated in MI, resulting in adverse ventricular remodeling marked by myocardial stiffening, thinning of the ventricular wall, and progressive heart failure. Consequently, the five-year mortality rate following MI remains unacceptably high, exceeding 50% in many populations. Given this pathophysiological complexity, current therapeutic strategies have shifted toward not only limiting acute injury but also promoting effective myocardial repair. Immunomodulatory interventions aimed at curbing excessive inflammation have demonstrated potential in halting ongoing damage[ 2 , 18 – 20 ]; however, they fall short in restoring contractile function or reversing structural deficits. In this context, therapeutic angiogenesis has emerged as a promising adjunctive strategy[ 21 , 22 ]. By stimulating the formation of new blood vessels within the ischemic and peri-infarct regions, angiogenesis enhances perfusion, supports the metabolic demands of surviving cardiomyocytes, and facilitates functional recovery of the damaged myocardium[ 23 – 25 ]. A comprehensive approach targeting oxidative stress, modulating inflammation, and promoting angiogenesis may offer synergistic benefits in attenuating post-MI remodeling and improving long-term cardiac outcomes. Haptoglobin (Hp), an acute phase protein, has the potential to be a therapeutic protein for MI due to its antioxidant[ 26 ], anti-inflammatory[ 27 ], immune-regulatory[ 28 ], and angiogenic properties. Hp's ability to scavenge free hemoglobin, limit ROS-mediated damage, modulate macrophage activity, and support neovascularization in ischemic tissue suggests it could be beneficial in treating MI[ 29 , 30 ]. We identified and cloned the Hp alpha-1 (Hp α1) chain as a therapeutic fragment, with the goal of developing a multifunctional functional peptide (MFP) capable of recapitulating the protective effects of full-length Hp in a more targeted and controlled manner[ 31 , 32 ]. Given its favorable biological properties and potential for synthetic production and modification, the MFP–derived peptide represents a novel therapeutic avenue for the treatment of MI, with the potential to simultaneously limit injury, modulate inflammation, and promote regeneration. This study explores the cardio protective potential of MFP in the context of MI, providing new insights into multifunctional protein-based strategies contribute to myocardial regeneration and functional restoration. Materials and Methods Cell Culture Human umbilical vein endothelial cells (HUVECs), H9c2 cardiomyoblast cells, and RAW264.7 murine macrophage cells were obtained from the Bioresource Collection and Research Center, Taiwan. HUVECs and H9c2 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA, Cat# 11995040), while RAW264.7 cells were cultured in RPMI 1640 medium (Gibco, USA, Cat# 11875085). All media were supplemented with 10% fetal bovine serum (FBS) (Gibco, USA, Cat# 26140079) and 1% antibiotic-antimycotic solution (Gibco, USA, Cat# 15240062). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air. MFP cloning, expression, purification and optimal MFP, modified from Hp α1 chain, was subcloned into a bacterial vector pET-30a(+) and this plasmid was transformed into E. coli strain BL21(DE3) competent cells. The His-tagged MFP was expressed by an E. coli expression system and purified by using immobilized metal affinity chromatography (MAM-50 His-NTA resin, EBL Biotechnology). Phosphate-buffered saline (PBS, pH 7.4) with 20 mM imidazole was used as binding buffer in this system, unbound proteins was washed out from the column by using PBS (pH 7.4) with 50 mM imidazole, and MFP was eluted by applying 50–500 mM imidazole gradient. The eluate fractions were resolved on 12% SDS-PAGE, the fractions including MFP were combined, concentrated, and dialyzed into 1x PBS (pH 7.4). Endotoxin was removed by using Pierce High-Capacity Endotoxin Removal Resin (Thermo scientific). In conclusion, more than 10 mg of > 95% pure proteins were obtained per liter of E. coli culture. MFP will be cloned and expressed by using an Escherichia coli (E. coli) expression system. The protein will be following purified by immobilized affinity chromatography. Antioxidant Activity Assay The antioxidant capacity of test compounds was evaluated using a thiobarbituric acid-reactive substances (TBARS) assay, which quantifies lipid peroxidation products. Cu²⁺ (copper sulfate) was used as an inducer of oxidative stress. Test substances included haptoglobin 1–1 (Hp 1–1) (Sigma-Aldrich), lysozyme, MFP, and probucol, which were each assessed across a range of concentrations (1.25 to 40 µM) using a 1:2 serial dilution scheme. In a typical assay, 4 µM CuSO4 (Sigma-Aldrich) and 40 mg of LDL were incubated with tested proteins. Following incubation with Cu²⁺ in the presence or absence of test proteins, the extent of lipid peroxidation was determined by measuring the formation of malondialdehyde (MDA)-TBA adducts spectrophotometrically. Antioxidant activity was quantified by the degree of inhibition of MDA formation compared to untreated controls. Cell Proliferation Assay The effect of MFP on endothelial cell viability and proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Human umbilical vein endothelial cells (HUVECs) were seeded at a density of 5,000 cells per well in 96-well plates (Falcon, Becton Dickinson, NJ) and incubated for 24 hours to allow cell attachment. Cells were then treated with MFP in conditioned media for 72 hours. After treatment, cells were washed with phosphate-buffered saline (PBS), and MTT solution (0.3 µg/µL in PBS; Sigma-Aldrich) was added to each well. Plates were incubated at 37°C for 4 hours to allow the formation of formazan crystals. Subsequently, 100 µL of dimethyl sulfoxide (DMSO) was added to dissolve the crystals, and absorbance was measured at 565 nm using a microplate reader. Each experimental condition was performed in sextuplicate (n = 6). In Vitro Wound Healing Assay (Scratch) An in vitro wound healing assay was performed to evaluate the effect of MFP on cell migration and wound closure under oxidative stress conditions. HUVECs were seeded into 6-well plates and cultured until reaching 95% confluence. A linear scratch was created across the cell monolayer using a sterile 200 µL pipette tip, followed by gentle washing with phosphate-buffered saline (PBS) to remove detached cells. To mimic oxidative injury, cells were treated with free hemoglobin (10 µM) as a source of reactive oxygen species. MFP was then applied at defined concentrations to assess its therapeutic effect on wound healing. Control and treatment groups were maintained in serum-free medium during the assay to exclude proliferation effects. Phase-contrast images of the wound area were captured at 0 h and 8 h using an inverted microscope. Wound closure was quantified by measuring the distance between the wound edges using ImageJ software. The percentage of wound closure was calculated as: [(initial wound width – final wound width) / initial wound width] × 100%. In Vitro Angiogenesis Assay To evaluate the pro-angiogenic effects of MFP, an in vitro capillary tube formation assay was performed using human umbilical vein endothelial cells (HUVECs) cultured on a growth factor-reduced extracellular matrix. Geltrex™ basement membrane matrix (growth factor reduced; Invitrogen) was used to simulate the extracellular environment. Approximately 200 µL of matrix was added to 9 mm diameter cell culture insert wells with 0.45 µm pore size (Millipore), and allowed to polymerize at 37°C for 30 minutes. HUVECs were resuspended in conditioned medium at a concentration of 1 × 10⁴ cells/mL, and seeded onto the polymerized matrix at a density of 5 × 10⁵ cells per well. Cells were incubated at 37°C for 16 hours to allow tube formation. After incubation, cells were stained with Calcein AM (Invitrogen) for 30 minutes to visualize live cells. Fluorescence images were captured using an Olympus BX51 fluorescence microscope. Three random microscopic fields per well were selected, and capillary-like structures were quantified by measuring the total tube length using ImageJ software. The assay was performed in triplicate for each condition. Rat Model of Myocardial Infarction and Treatment Protocol Rat Model of Myocardial Infarction and Treatment Protocol Adult male Sprague-Dawley (SD) rats (250 ± 20 g), obtained from LASCO (Taipei, Taiwan), were used to establish a chronic myocardial infarction (MI) model. MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery, as previously described. Successful LAD occlusion was confirmed intraoperatively by the presence of ST-segment elevation on electrocardiography (ECG) and visible myocardial surface cyanosis distal to the ligation site. Sham-operated animals underwent the same surgical procedure without LAD ligation. Following surgery, LAD-ligated rats were randomly assigned to two groups: (1) saline control and (2) MFP treatment. In the MFP group, intramyocardial injections of MFP (1 µg/mL) were administered at five distinct sites within the peri-infarct (border) zone. Rats in the control and sham groups received an equivalent volume of sterile saline. All animals were monitored for four weeks post-MI. All experimental procedures were conducted in accordance with the guidelines of the Animal Ethics Committee of Taipei Medical University and were approved prior to initiation of the study. Echocardiography Transthoracic echocardiography was performed to assess cardiac structure and function using a CX50 ultrasound system (Philips Ultrasound System, Andover, MA, USA). Rats were anesthetized with light isoflurane inhalation to maintain spontaneous breathing and minimize cardiac depression. The parasternal short-axis view of the left ventricle (LV) was obtained, and M-mode recordings were used to evaluate ventricular wall motion and chamber dimensions. The left ventricular internal diameter at end-diastole (LVIDd) and at end-systole (LVIDs) were measured. Left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were calculated using standard formulas: LVEF (%) = [(LVIDd³ – LVIDs³) / LVIDd³] × 100 LVFS (%) = [(LVIDd – LVIDs) / LVIDd] × 100 All measurements were performed in triplicate from three consecutive cardiac cycles and analyzed in a blinded manner to ensure objectivity. Histological and TUNEL Analysis Paraffin-embedded heart tissue sections (4 µm thick) were prepared for histological and apoptosis evaluation. Sections were deparaffinized in xylene and rehydrated through a graded ethanol series (100%, 90%, 70%) followed by a final rinse in distilled water. For general histology and fibrosis assessment, sections were stained with hematoxylin and eosin (H&E) or Masson’s Trichrome. In Masson’s Trichrome-stained sections, collagen fibers appeared blue, myocardial muscle fibers stained red, and nuclei stained blue-black, enabling clear delineation of fibrotic and viable tissue. To detect apoptotic cells, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using a commercial apoptosis detection kit (Roche, Branchburg, NJ, USA), following the manufacturer’s instructions. TUNEL-positive nuclei were visualized by fluorescence microscopy. The percentage of apoptotic cells was quantified by calculating the ratio of TUNEL-positive nuclei to total nuclei in randomly selected fields. Immunohistochemistry Immunohistochemical analysis was performed on paraffin-embedded heart tissue sections to assess the expression of vascular, inflammatory, and immune cell markers. Sections (4 µm thick) were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval. Endogenous peroxidase activity was quenched using hydrogen peroxide. After rinsing, sections were blocked with 5% bovine serum albumin (BSA) to reduce nonspecific binding. Tissues were incubated overnight at 4°C with primary antibodies specific to the following markers: CD31 (1:2000; Abcam, Cat# ab182981), VEGFR (1:800; Abcam, Cat# ab9698), CD68 (1:100; Abcam, Cat# ab31630), CD163 (1:1000; Proteintech, Cat# 16646-1-AP), IL-6 (1:50; GeneTex, Cat# GTX17623), Control IgG (1:100; Abcam, Cat# ab37415) Following primary antibody incubation, sections were treated with an HRP-conjugated secondary antibody and developed using 3,3'-diaminobenzidine (DAB) as the chromogen. Slides were then counterstained with hematoxylin, dehydrated, and mounted. Immunoreactive staining was visualized and evaluated under a light microscope. Flow Cytometry Analysis of Mitochondrial Membrane Potential and Apoptosis Mitochondrial membrane potential (ΔΨm) was assessed using the JC-1 assay kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. JC-1 dye selectively accumulates in mitochondria, forming red-fluorescent J-aggregates in cells with high ΔΨm, while remaining as green-fluorescent monomers in cells with depolarized (low ΔΨm) mitochondria. After experimental treatment, cells were incubated with JC-1 working solution at 37°C for 20 minutes in the dark, washed twice with dye buffer, and immediately analyzed by flow cytometry. The ratio of red (aggregates) to green (monomers) fluorescence intensity was used to quantify changes in ΔΨm. In parallel, apoptosis was assessed using Annexin V-FITC and propidium iodide (PI) staining (Invitrogen). Cells treated with hydrogen peroxide (H₂O₂), with or without MFP, were resuspended in binding buffer and incubated with Annexin V-FITC and PI for 15 minutes at room temperature in the dark. Samples were analyzed by flow cytometry to distinguish viable, early apoptotic, late apoptotic, and necrotic cell populations. Mitochondrial Superoxide Detection Mitochondrial superoxide production was assessed using MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen), following the manufacturer’s protocol. Cells were incubated with 2 µM MitoSOX Red reagent at 37°C for 30 minutes in the dark. After incubation, cells were washed with phosphate-buffered saline (PBS) to remove excess dye, and red fluorescence, indicative of mitochondrial superoxide accumulation, was visualized using a fluorescence microscope. As a positive control for mitochondrial dysfunction, cells were treated with 2 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 30 minutes prior to MitoSOX staining to induce mitochondrial depolarization and elevated superoxide generation. Quantitative Reverse Transcription PCR (qRT-PCR) Total RNA was extracted from cells or tissues using TRIzol™ reagent (Invitrogen), following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using SYBR® Premix Ex Taq™ II (Takara Bio) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Gene expression levels were normalized to the housekeeping gene GAPDH, and relative expression was calculated using the 2^–ΔΔCt method. The primer sequences used were as follows: CD163: (Forward: 5′-GGCTAGACG AAGTCATCTGCAC-3′, Reverse: 5′-CTTCGTTGGTCAGCCTCAGAGA-3′), IL-6: (Forward: 5′-TACCACTTCACAAGTCGGAGGC-3′, Reverse: 5′-CTGCAAGTG CATCATCGTTGTTC-3′), IL-10: (Forward: 5′-CGGGAAGACAATAACTGCACC C-3′, Reverse: 5′-CGGTTAGCAGTATGTTGTCCAGC-3′), IL-1β: (Forward: 5′-TGGACCTTCCAGGATGAGGACA-3′, Reverse: 5′-GTTCATCTCGGAGCCTGT AGTG-3′), GAPDH: (Forward: 5′-TCACCACCATGGAGAAGGC-3′, Reverse: 5′-GCTAAGCAGTTGGTGGTGCA-3′) All reactions were run in triplicate, and melting curve analysis was conducted to ensure amplification specificity. Western Blotting Total protein was extracted from cultured cells using RIPA lysis buffer supplemented with protease inhibitors (Thermo Scientific), followed by centrifugation at 14,000 × g for 15 minutes at 4°C to remove debris. Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (20–30 µg per sample) were separated via SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with 5% non-fat milk in TBS-T buffer (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature and incubated overnight at 4°C with the following primary antibodies: cleaved Caspase-3 (1:1000, Cell Signaling Technology, Cat# 9664), Bax (1:1000, Cell Signaling Technology, Cat# 2772), and Bcl-2 (1:1000, GeneTex, Cat# GTX100064). After washing, membranes were incubated with species-appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection substrate (Millipore) and imaged using a UVP BioImaging System. β-actin was used as a loading control. RNA Sequencing and Bioinformatics Network Analysis Total RNA was isolated and quantified, and high-quality RNA (1 µg) was used for library preparation using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA), following the manufacturer's protocol. Polyadenylated mRNA was enriched using oligo(dT)-conjugated magnetic beads and subsequently fragmented at elevated temperatures. First-strand cDNA synthesis was performed using random hexamer primers and reverse transcriptase, followed by second-strand synthesis to generate double-stranded cDNA. After end repair, 3’-adenylation, and adaptor ligation, cDNA fragments were purified using the AMPure XP system (Beckman Coulter, Brea, CA, USA). Library quality and insert size distribution were assessed using the Agilent 2100 Bioanalyzer, and concentrations were validated using quantitative real-time PCR. Paired-end sequencing (150 bp reads) was carried out on an Illumina NovaSeq 6000 platform by Genomics, BioSci & Tech Co. (New Taipei City, Taiwan). For transcriptome analysis, raw sequencing reads were quality-filtered and aligned to the reference genome. Differentially expressed genes were identified and analyzed using Ingenuity Pathway Analysis (IPA, Qiagen, Redwood City, CA, USA) to elucidate regulatory networks, canonical signaling pathways, and functional gene associations in response to MFP treatment. Statistical Analysis All data are expressed as mean ± standard error of the mean (SEM). Each experiment was independently repeated at least three times to ensure reproducibility. Statistical analyses were conducted using GraphPad Prism software (version 5.0; GraphPad Software, San Diego, CA, USA). For comparisons among multiple groups, one-way analysis of variance (ANOVA) was employed. Post hoc analyses were performed using Tukey’s multiple comparisons test for data with equal variances or the Dunnett’s T3 test when unequal variances were detected. All statistical tests were two-tailed, and a p-value < 0.05 was considered statistically significant. Results MFP expression and purification To produce recombinant MFP protein, the MFP coding sequence was subcloned into the pET-30a (+) bacterial expression vector, which contains a T7 promoter and His-tag sequences for affinity purification (Figure S1A). The resulting plasmid was transformed into E. coli BL21(DE3) cells. Protein expression was induced, and His-tagged MFP was purified using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin under native conditions. The purification process involved binding in phosphate-buffered saline (PBS, pH 7.4) containing 20 mM imidazole, followed by a wash step with 50 mM imidazole to remove nonspecifically bound proteins. MFP was eluted using a linear imidazole gradient (50–500 mM). Eluted fractions were analyzed by 12% SDS-PAGE stained with Coomassie blue (Figure S1B). MFP appeared as a prominent band at the expected molecular weight (~ 25 kDa), with peak enrichment observed in fractions 6–10. These fractions were pooled, concentrated, and dialyzed into PBS (pH 7.4). Following endotoxin removal, the final preparation yielded more than 10 mg of > 95% pure MFP per liter of bacterial culture. These results confirm the robust expression and high-yield purification of recombinant MFP suitable for downstream functional and therapeutic studies. MFP Exhibits Potent Antioxidant Activity Against Cu²⁺-Induced Lipid Peroxidation Oxidative stress is a well-established contributor to myocardial injury following acute myocardial infarction (AMI), largely through increased lipid peroxidation and inflammation[ 33 ]. To assess the antioxidant capacity of MFP, a copper ion (Cu²⁺)-induced low-density lipoprotein (LDL) peroxidation assay was employed. The extent of lipid peroxidation was measured via malondialdehyde (MDA) formation using a TBARS-based method. As shown in Fig. 1 A, MFP demonstrated a strong, dose-dependent inhibition of MDA formation, indicating a robust antioxidant effect. Compared with known antioxidants, MFP was significantly more potent than both native Haptoglobin 1–1 (Hp 1–1) and probucol, an established lipid-lowering agent with antioxidant properties[ 26 ]. While Hp 1–1 and probucol exhibited moderate suppression of MDA production at higher concentrations, MFP nearly abolished MDA formation at concentrations as low as 10–20 µM. In contrast, lysozyme, used as a negative control protein, showed no appreciable antioxidant activity across the tested concentration range. These findings identify MFP as a highly effective antioxidant peptide, superior both endogenous Hp 1–1 and pharmacological probucol in this in vitro model. Given the importance of oxidative injury in post-MI pathology, MFP may represent a promising candidate for therapeutic intervention targeting oxidative stress in AMI. MFP Enhances Endothelial Cell Proliferation, Migration, and Angiogenesis in Vitro Endothelial repair and angiogenesis are critical for tissue regeneration following acute myocardial infarction (AMI)[ 34 ]. Given the clinical relevance of vascular endothelial growth factor (VEGF) elevation post-AMI, we investigated whether MFP promotes endothelial cell function under oxidative stress conditions. To assess endothelial proliferation, human umbilical vein endothelial cells (HUVECs) were cultured in high glucose medium and treated with increasing concentrations of MFP. A dose-dependent increase in cell proliferation was observed, with a peak proliferative response (~ 3-fold) at 0.125 mg/mL MFP (Fig. 1 B). Higher concentrations reduced this effect, suggesting an optimal therapeutic window. Next, we evaluated endothelial migration using a scratch wound healing assay in the presence of hemoglobin (Hb), a known inducer of oxidative stress via reactive oxygen species (ROS) production[ 35 ]. Hb significantly impaired wound closure, whereas MFP supplementation reversed this inhibitory effect. By 8 hours, MFP-treated HUVECs achieving approximately 70% wound closure, compared with in the Hb-alone group (Fig. 1 C). To assess angiogenic capacity, HUVECs were seeded on growth factor-depleted Matrigel to evaluate tube formation. MFP treatment robustly promoted angiogenesis, as evidenced by enhanced tube length and branch point formation. Notably, the angiogenic effect of MFP was comparable to that of VEGF, a key regulator of neovascularization [ 36 ](Fig. 1 D). Collectively, these results demonstrate that MFP promotes endothelial proliferation, restores cell migration impaired by oxidative stress, and induces angiogenesis, underscoring its potential as a vascular regenerative therapy post-AMI. MFP Promotes Endothelial Preserves Cardiac Function Following Myocardial Infarction in vivo Echocardiographic analysis was performed 14 days after MI induction to assess cardiac function[ 37 ]. M-mode imaging revealed marked systolic dysfunction in MI animals, as shown by reduced left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), and increased left ventricular internal diameters during systole (LVIDs) and diastole (LVIDd) (Fig. 2 A). Notably, MFP administration significantly preserved LVEF and LVFS and attenuated ventricular dilation compared to MI alone (Fig. 2 B). Together, these findings suggest that MFP preserves cardiac structure and function in vivo after MI, highlighting its therapeutic potential for post-infarction remodeling and recovery. MFP Attenuates Myocardial Fibrosis Post-Infarction Myocardial fibrosis is a hallmark of pathological cardiac remodeling following myocardial infarction (MI), contributing to impaired ventricular compliance and progression to heart failure[ 9 , 38 ]. To assess whether MFP confers anti-fibrotic effects in vivo, histological and molecular analyses were conducted on cardiac tissues harvested four weeks after left anterior descending (LAD) artery ligation. Masson's trichrome staining revealed extensive collagen deposition and wall thinning in the infarcted left ventricle of MI animals, indicative of substantial myocardial fibrosis (Fig. 3 A). In contrast, MFP-treated animals exhibited markedly reduced fibrotic area and preserved myocardial structure. Quantitative analysis confirmed a significant reduction in interstitial collagen content in the MFP group compared to the untreated MI group (p < 0.01; Fig. 3 B). Hematoxylin and eosin (H&E) staining further demonstrated disorganized myocardial architecture and fibrotic infiltration in MI hearts, which were notably alleviated by MFP treatment (Fig. 3 C). Immunohistochemical staining for transforming growth factor-beta 1 (TGF-β1), a key mediator of fibrotic signaling, showed strong upregulation in the infarct region of MI hearts. MFP administration significantly suppressed TGF-β1 expression and reduced infarct size (Fig. 3 D), supporting a direct anti-fibrotic mechanism. Together, these data indicate that MFP mitigates adverse fibrotic remodeling post-MI, potentially contributing to improved cardiac structure and function. MFP Suppresses Post-Infarction Inflammatory Response via IL-6 Downregulation Excessive inflammation following myocardial infarction (MI) contributes to adverse cardiac remodeling and impaired recovery[ 39 ]. To investigate the anti-inflammatory effects of MFP, we evaluated interleukin-6 (IL-6) expression in infarcted cardiac tissue using immunohistochemistry. As shown in Fig. 3 E, IL-6 expression was markedly elevated in the myocardium of MI animals compared to controls, consistent with an acute inflammatory response. Treatment with MFP significantly reduced IL-6 immunoreactivity in the infarct and peri-infarct regions, indicating attenuation of the inflammatory response. Quantification of IL-6-positive staining revealed a dramatic decrease in the MFP-treated group compared to the MI group (p < 0.01), suggesting that MFP mitigates inflammatory signaling after cardiac injury. These results highlight MFP’s immunomodulatory role in the post-MI setting, supporting its potential to limit inflammation-driven myocardial damage. MFP Attenuates Cardiomyocyte Apoptosis After Myocardial Infarction by Counteracting Oxidative Stress Loss of cardiomyocytes is a key driver of adverse left ventricular remodeling and subsequent cardiac dysfunction following myocardial infarction[ 40 ] (MI). Oxidative stress, particularly the overproduction of reactive oxygen species (ROS), plays a critical role in promoting cardiomyocyte apoptosis in the ischemic myocardium[ 41 ]. To investigate the cardioprotective effect of MFP, we assessed both in vivo and in vitro markers of apoptosis. TUNEL staining revealed a substantial increase in apoptotic cardiomyocytes in the infarct border zone of MI hearts compared to the sham-operated group (Fig. 4 A–B), consistent with significant ischemia-induced cell death. Notably, MFP treatment significantly reduced the number of TUNEL-positive nuclei, indicating decreased apoptosis in the infarcted myocardium. To further explore the anti-apoptotic effect of MFP under oxidative stress, H9c2 cardiomyoblasts were exposed to hydrogen peroxide (H₂O₂, 600 µM) in vitro. Flow cytometric analysis with Annexin V/propidium iodide staining demonstrated a marked increase in apoptotic cells following H₂O₂ treatment (73.2% ± 3%) compared to control (23.97%). Pre-treatment with MFP significantly attenuated H₂O₂-induced apoptosis (Fig. 4 D–E). Western blot analysis of apoptosis-related proteins corroborated these findings. H₂O₂ exposure led to increased expression of pro-apoptotic markers Bax and cleaved caspase-3, alongside decreased expression of the anti-apoptotic protein Bcl-2 (Fig. 4 C). MFP treatment reversed these trends, upregulating Bcl-2 while suppressing Bax and cleaved caspase-3 expression. Collectively, these results suggest that MFP promotes cardiomyocyte survival following MI by counteracting oxidative stress-induced apoptosis and modulating key apoptotic signaling pathways. MFP Attenuates Oxidative Stress and Preserves Mitochondrial Function Following Myocardial Infarction Oxidative stress is a major contributor to myocardial injury following infarction, promoting mitochondrial dysfunction and cardiomyocyte apoptosis[ 42 ]. To evaluate the antioxidative capacity of MFP in vivo, we quantified superoxide dismutase (SOD) activity and MDA levels in myocardial tissue[ 43 ]. Myocardial infarction significantly reduced SOD activity compared to the sham group; notably, MFP administration restored SOD levels toward baseline (Fig. 5 A). In parallel, MDA—an established marker of lipid peroxidation—was markedly elevated in the MI group, whereas MFP treatment significantly decreased MDA levels, indicating reduced oxidative damage. To explore MFP’s role in mitigating mitochondrial oxidative stress, we assessed mitochondrial superoxide generation using mitoSOX Red staining in H9c2 cardiomyoblasts. Exposure to hydrogen peroxide (H₂O₂) induced a substantial increase in mitochondrial ROS, evidenced by enhanced mitoSOX fluorescence. MFP pretreatment significantly suppressed H₂O₂-induced mitochondrial superoxide accumulation (Fig. 5 B), suggesting a protective effect on mitochondrial redox balance. Given the importance of mitochondrial integrity in cell survival, we further evaluated mitochondrial membrane potential (ΔΨm) using JC-1 staining. H₂O₂ exposure led to a significant increase in the green/red fluorescence ratio, reflecting ΔΨm dissipation and mitochondrial depolarization (Fig. 5 C–D). Remarkably, MFP treatment preserved ΔΨm by reducing the green/red fluorescence ratio, indicative of improved mitochondrial membrane stability. These findings demonstrate that MFP effectively counteracts oxidative stress and preserves mitochondrial function in cardiomyocytes, supporting its therapeutic potential in the context of myocardial infarction. MFP Enhances Angiogenesis and Coronary Revascularization Following Myocardial Infarction Effective neovascularization is critical for myocardial repair and functional recovery following infarction[ 23 , 44 ]. To determine whether MFP facilitates post-MI angiogenesis, we examined endothelial cell proliferation and vascular regeneration in infarcted myocardium via immunohistochemical analysis. CD31, an established endothelial marker, was used to assess capillary density in myocardial cryosections. Compared with the sham group, CD31 expression was markedly diminished in the MI group, indicating compromised microvascular integrity (Fig. 6 ). Notably, treatment with MFP significantly restored CD31 expression, suggesting enhanced endothelial proliferation and neovascularization in the infarcted region. In addition, we assessed the expression of vascular endothelial growth factor receptor (VEGFR), a key mediator of angiogenic signaling. VEGFR levels were markedly elevated in MFP-treated hearts compared to both the MI and control groups, indicating potentiation of endogenous pro-angiogenic pathways. Collectively, these findings demonstrate that MFP promotes angiogenesis in the infarcted myocardium, likely through upregulation of CD31 and VEGFR expression. This angiogenic activity may contribute to improved myocardial perfusion, reduced tissue injury, and enhanced cardiac repair post-infarction. MFP Attenuates Inflammatory Cell Infiltration and Promotes Reparative Macrophage Polarization Following Myocardial Infarction Post-infarction inflammation is a key driver of adverse cardiac remodeling and heart failure progression[ 20 , 39 ]. To evaluate the impact of MFP on inflammatory responses in the myocardium, immunohistochemical staining was performed to identify total macrophage infiltration (CD68), pro-inflammatory M1 macrophages (CD86), and anti-inflammatory M2 macrophages (CD206). As shown in Fig. 7 A, myocardial tissues from the MI group displayed a substantial increase in CD68⁺ and CD86⁺ macrophages, indicative of pronounced inflammatory cell recruitment and M1 polarization. Strikingly, treatment with MFP significantly reduced both CD68⁺ and CD86⁺ macrophage infiltration, suggesting effective suppression of pro-inflammatory responses. In contrast, CD206⁺ M2 macrophages were sparsely observed in infarcted myocardium, whereas MFP administration markedly increased CD206 expression, denoting a phenotypic switch towards reparative, anti-inflammatory macrophages. This shift in macrophage polarization toward the M2 lineage is associated with enhanced tissue repair, resolution of inflammation, and improved cardiac recovery. To further confirm the direct modulatory effect of MFP on macrophage polarization, Raw 264.7 macrophages were treated in vitro with MFP for 24 hours. Flow cytometric analysis revealed significantly elevated surface expression of CD206 and CD11b in MFP-treated cells, consistent with M2 polarization (Fig. 7 B). Gene expression profiling further supported this shift, showing increased mRNA levels of CD163, IL-6, and IL-10—markers associated with anti-inflammatory M2 function—while expression of the pro-inflammatory cytokine IL-1β was markedly downregulated (Fig. 7 C). Collectively, these findings underscore the immunomodulatory potential of MFP in the post-MI setting. By reducing inflammatory macrophage infiltration and promoting M2 macrophage reprogramming, MFP not only mitigates inflammation but also fosters a reparative microenvironment conducive to myocardial healing. This dual anti-inflammatory and pro-reparative action positions MFP as a promising candidate for therapeutic intervention in post-infarction cardiac remodeling. Transcriptomic Profiling Reveals MFP Modulates Cardioprotective and Immunoregulatory Pathways in Oxidative-Stressed Cardiomyocytes To gain mechanistic insight into the cardioprotective effects of MFP at the transcriptomic level, RNA-sequencing was performed on H9c2 cardiomyocytes exposed to oxidative stress (600 µM H₂O₂) with or without MFP treatment. A total of 860 differentially expressed genes (DEGs) were identified between the MFP- and PBS-treated groups, including 361 significantly upregulated and 499 downregulated genes (Log₂FC > 0.58 or < − 0.58, adjusted p < 0.05) (Fig. 8 A). These DEGs reflect MFP’s capacity to reprogram the stress-induced transcriptomic landscape of cardiomyocytes. Ingenuity Pathway Analysis (IPA) of the DEGs revealed robust modulation of key canonical signaling pathways involved in inflammation, oxidative stress, cell survival, and repair. The top 10 significantly enriched canonical pathways in the MFP group included upstream regulators of cytokine signaling, mitochondrial homeostasis, and pro-survival cascades (Fig. 8 B). Importantly, IPA’s "Tox Function" analysis indicated marked suppression of molecular signatures linked to pathological cardiac remodeling. MFP treatment led to a substantial reduction in pathways associated with cardiac dysfunction, including ventricular dilation, systolic dysfunction, cardiomyocyte necrosis, and fibrosis (Fig. 8 C). Furthermore, IPA identified a set of 12 MFP-regulated genes associated with M2 macrophage polarization, including CD163, IL10, and MRC1, providing molecular corroboration for the observed shift toward an anti-inflammatory reparative immune environment (Fig. 8 D). Together, these transcriptomic findings suggest that MFP exerts its cardioprotective effects through a multifactorial mechanism involving modulation of redox-sensitive gene networks, suppression of deleterious cardiac stress pathways, and promotion of immunoresolving phenotypes. These data provide strong molecular evidence supporting MFP’s therapeutic potential in mitigating post-MI myocardial damage and maladaptive remodeling. Discussion Despite significant advancements in reperfusion strategies and pharmacological therapies, myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide. While current treatment paradigms focus primarily on restoring coronary perfusion and mitigating acute complications, they often fail to address the complex cellular and molecular cascades triggered by ischemia-reperfusion injury—namely oxidative stress, inflammation, endothelial dysfunction, and maladaptive ventricular remodeling[ 45 , 46 ]. Thus, there is an urgent need for novel therapies that not only limit initial injury but also promote myocardial repair and functional recovery. In this context, our study identifies MFP, a multifunctional therapeutic peptide, as a promising cardioprotective agent with significant translational relevance. We demonstrate that MFP exerts its effects through a multifaceted mechanism of action encompassing antioxidant activity, anti-apoptotic signaling, immunomodulation, and promotion of angiogenesis, ultimately culminating in improved cardiac structure and function post-MI. Anti-Remodeling and Anti-Fibrotic Effects Left ventricular (LV) remodeling following MI is a pathophysiological continuum that encompasses infarct expansion, cardiomyocyte hypertrophy, and progressive interstitial fibrosis[ 9 ]—all of which contribute to systolic dysfunction and heart failure. Our histological and molecular data confirm that MFP significantly attenuates myocardial interstitial fibrosis and collagen deposition. Mechanistically, this effect is associated with downregulation of TGF-β1, a key profibrotic cytokine known to drive fibroblast-to-myofibroblast differentiation and extracellular matrix accumulation. Additionally, MFP reduced the differentiates collagen expression (Fig. 3 B), further supporting its role in preserving myocardial architecture and mitigating adverse LV remodeling. Anti-Apoptotic and Antioxidant Activity Cardiomyocyte apoptosis is a hallmark of ischemia-reperfusion injury and contributes to progressive loss of viable myocardium post-infarction[ 9 ]. Our study shows that MFP treatment significantly reduced TUNEL-positive nuclei and suppressed pro-apoptotic markers Bax and cleaved caspase-3, while maintaining expression of the anti-apoptotic gene Bcl-2. These changes were accompanied by enhanced activation of the PI3K/Akt signaling pathway, which is well-documented for its cytoprotective role in the heart. MFP also conferred robust antioxidant protection, as evidenced by decreased MDA levels and enhanced SOD activity[ 41 ]. Preservation of mitochondrial membrane potential in MFP-treated hearts suggests that mitochondrial integrity is maintained, likely contributing to improved cardiomyocyte survival. Transcriptomic analysis further reinforced these findings by revealing downregulation of oxidative stress-associated genes and suppression of cardiac dysfunction-related “Tox Functions,” including ventricular dilation, necrosis, and systolic failure. Pro-Angiogenic and Endothelial Protective Effects Neovascularization within the infarct and peri-infarct regions is critical for restoring tissue perfusion and supporting myocardial regeneration[ 23 , 34 , 44 , 47 ]. MFP promoted endothelial cell proliferation, migration, and tube formation in vitro, with efficacy comparable to that of VEGF. In vivo, this translated into increased capillary density and elevated VEGFR expression in the infarct border zone. These findings suggest that MFP enhances tissue revascularization and oxygen delivery, which are essential for optimal healing and functional recovery. Immune Modulation and Macrophage Polarization Unresolved inflammation following MI exacerbates tissue damage and impairs repair[ 20 , 39 ]. One of the most compelling aspects of MFP's action lies in its immunomodulatory capability. Our in vivo and in vitro data indicate that MFP shifts macrophage polarization from a pro-inflammatory M1 phenotype to a reparative M2 phenotype, as evidenced by reduced IL-6 expression, decreased M1 macrophage infiltration, and upregulation of M2 markers such as CD163, IL-10, and MRC1. This polarization shift likely involves multiple converging signaling pathways. For instance, activation of PI3K/Akt not only supports cardiomyocyte survival but also promotes M2 macrophage programming[ 27 , 28 ]. In parallel, modulation of PPAR, LXR/RXR, and Notch/TLR4 pathways may contribute to the observed immune reprogramming. Additionally, MFP may influence regulatory T-cell (Treg) activity and downstream cytokine cascades (e.g., IL-4, IL-10), which are known to support M2 polarization and angiogenesis via VEGF-A production. These findings highlight the intricate interplay between MFP and the immune system, underscoring its potential to modulate inflammation-resolving processes critical for cardiac repair. Transcriptomic Insights and Systems-Level Mechanisms RNA sequencing analysis identified 860 differentially expressed genes associated with MFP treatment, implicating critical pathways related to inflammation, oxidative injury, fibrosis, and immune regulation. Importantly, Ingenuity Pathway Analysis (IPA) predicted suppression of adverse cardiac phenotypes and activation of pathways involved in tissue repair. These systems-level insights provide a molecular foundation for the observed histological and functional improvements and underscore MFP's ability to orchestrate complex reparative responses in the infarcted myocardium. MFP as a Multi-Phase Modulator of Post-MI Cardiac Repair Cardiac repair after myocardial infarction progresses through three interrelated phases[ 48 ]—inflammatory, proliferative, and maturation—each offering distinct therapeutic targets. MFP demonstrates a unique capacity to beneficially modulate all three stages. In the inflammatory phase (days 1–4), MFP’s potent ROS-scavenging and anti-inflammatory properties attenuate oxidative stress and excessive immune activation, limiting collateral tissue damage and preserving viable myocardium. During the proliferative phase (days 4–14), MFP enhances endothelial cell proliferation, accelerates endothelial wound closure, and promotes angiogenesis, improving perfusion to the peri-infarct zone while modulating macrophage polarization toward a reparative M2 phenotype. In the maturation phase (beyond day 14), MFP’s anti-fibrotic effects limit maladaptive extracellular matrix deposition, reduce scar stiffness, and preserve left ventricular compliance, thereby mitigating adverse remodeling. By simultaneously targeting oxidative stress, inflammation, neovascularization, and fibrosis in a temporally coordinated manner, MFP addresses the complex, sequential biology of post-MI repair with a single therapeutic agent—an approach with clear translational promise for improving long-term cardiac outcomes. Three-phase cardioprotective actions of MFP after acute myocardial infarction The therapeutic actions of the multifunctional peptide (MFP) across the three sequential phases of post–acute myocardial infarction (AMI) repair (Fig. 9 ). In the inflammatory phase (days 1–4), MFP mitigates oxidative stress and suppresses inflammation through reactive oxygen species scavenging. In the proliferative phase (days 4–14), MFP supports pro-angiogenic endothelial cell activity and promotes tissue regeneration. In the maturation phase (beyond day 14), MFP exerts anti-fibrotic effects by reducing extracellular matrix deposition and enhancing reparative M2 macrophage polarization, thereby attenuating scar formation and improving myocardial remodeling[ 8 , 49 ]. From a therapeutic perspective, MFP offers several advantages: high-yield production, biological stability, and multimodal efficacy. Unlike conventional agents that typically target singular pathological mechanisms, MFP’s broad-spectrum activity addresses the multifactorial nature of MI pathology, potentially filling a major gap in the current treatment paradigm. The observed improvements in cardiac function, structure, and cellular viability post-MI suggest that MFP could serve as a first-in-class biologic for cardiac regeneration. Conclusion Our study provides compelling preclinical evidence that MFP is a potent and multifunctional therapeutic candidate capable of attenuating ischemic injury and enhancing myocardial repair following MI. Through coordinated modulation of fibrosis, apoptosis, oxidative stress, angiogenesis, and immune responses, MFP offers a promising avenue for regenerative cardiology. Future studies will be required to evaluate its long-term safety, pharmacokinetics, and therapeutic efficacy in large animal models and clinical trials. Nevertheless, these findings establish a strong foundation for the translational development of MFP and its potential to significantly impact the management of ischemic heart disease. Abbreviations MI Myocardial infarction ROS Reactive oxygen species MFP Multifunctional functional peptide HUVECs Human umbilical vein endothelial cells LAD Left anterior descending LV Left ventricular LVIDd Left ventricular internal diameter at end-diastole LVIDs Left ventricular internal diameter at end-systole LVEF Left ventricular ejection fraction LVFS Left ventricular fractional shortening ANOVA One-way analysis of variance MDA Malondialdehyde SOD Superoxide dismutase Declarations Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/sXXXX-XXX-XXXXX-X.. Acknowledgements The authors gratefully acknowledge the technical support provided by the Core Facility Center and Laboratory Animal Center of Taipei Medical University. Author contributions HYL, HYC, conducted experiments and acquired the data. WCC data statistic. CWC, CYC, and CP MFP purification. CHH MFP large scale production. FLM, KW, CHC, TRK and ATHW experiment design and paper write consult. CYH and CMS animal experiment and echocardiograms obtained. CCS and TMC wrote the manuscript and provided funding. All authors read and approved the final manuscript. Funding This work was financially supported by the National Science and Technology Council (NSTC), Taiwan (Grant No. NSTC 114-2314-B-038-091-MY3 to Tsai-Mu Cheng; NSTC 113-2314-B-038-010 and NSTC114-2314-B-038-158 to Alexander T.H. Wu; NSTC 113-2314-B-038-008 and NSTC 114-2314-B-038-156 to Chun-Ming Shih). Taipei Medical University, and the Higher Education Sprout Project by the Ministry of Education (DP2-TMU-114-O-01), Taiwan. Taipei Medical University-National Defense Medical Center Joint Research Program (TMU-NDMC-11304) Data availability This published article and its supplementary information files include all data generated or analyzed during this study. Ethics approval and consent to participate All experimental procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University and were approved prior to initiation of the study. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests References Townsend N, Wilson L, Bhatnagar P, Wickramasinghe K, Rayner M, Nichols M. Cardiovascular disease in Europe: epidemiological update 2016. Eur Heart J. 2016;37(42):3232–45. 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Selectins and Immune Cells in Acute Myocardial Infarction and Post-infarction Ventricular Remodeling: Pathophysiology and Novel Treatments. Front Immunol. 2019;10:300. Additional Declarations No competing interests reported. Supplementary Files FigureS1.docx Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Compared with native haptoglobin 1-1 (Hp 1-1) and the antioxidant drug probucol, MFP demonstrates superior efficacy, nearly abolishing MDA production at low micromolar concentrations. Lysozyme served as a negative control. (B) MFP enhances proliferation of human umbilical vein endothelial cells (HUVECs) cultured under high-glucose stress, with an optimal proliferative effect observed at 0.125 mg/mL. (C) In a scratch wound assay, hemoglobin (Hb)-induced impairment of endothelial migration is rescued by MFP, achieving ~70% wound closure within 8 h. (D) Tube formation assay on growth factor-depleted Matrigel demonstrates that MFP promotes angiogenesis, with tube length and branching comparable to VEGF.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8334699/v1/63f47c721b690f63bd96072e.png"},{"id":98195827,"identity":"cf9f1e15-bdd4-45c2-b29b-7c68e7c2ed2f","added_by":"auto","created_at":"2025-12-15 06:41:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":671589,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMFP Preserves Left Ventricular Function and Limits Remodeling in a Rat Model of Myocardial Infarction. \u003c/strong\u003eMFP treatment maintains systolic performance and attenuates ventricular dilation following myocardial infarction (MI). (A) Representative M-mode echocardiograms obtained 14 days post-MI demonstrate marked systolic impairment in untreated MI rats, which is mitigated by MFP administration. (B) Quantitative echocardiographic parameters: left ventricular end-diastolic dimension (LVEDD) and end-systolic dimension (LVESD) were measured; left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were calculated. Data are presented as mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. MI group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8334699/v1/6c26aa0dfff44b14838c5d84.png"},{"id":98195819,"identity":"7f6c17da-bb60-4e0d-b7a6-1ebc31fa9f08","added_by":"auto","created_at":"2025-12-15 06:41:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1715973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMFP attenuates adverse post-infarction remodeling by reducing myocardial fibrosis and suppressing inflammatory signaling. \u003c/strong\u003e(A) Representative Masson’s trichrome–stained sections showing collagen deposition (blue) and wall thinning in infarcted left ventricles. MFP treatment markedly reduced fibrotic area compared with MI alone. (B) Quantification of interstitial collagen fraction (% area) confirming significant fibrosis reduction in the MFP group. (C) Hematoxylin and eosin (H\u0026amp;E) staining illustrating preserved myocardial architecture and reduced fibrotic infiltration with MFP therapy. (D) Immunohistochemical staining of TGF-β1 in infarct regions showing robust upregulation in MI hearts and significant suppression following MFP treatment. (E) Immunohistochemistry for IL-6 demonstrating elevated inflammatory response in MI hearts, attenuated by MFP administration in both infarct and peri-infarct zones. Quantitative data are presented as mean ± SEM. **p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus MI group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8334699/v1/1c6885bcf5cdd112cfda2f3f.png"},{"id":98195852,"identity":"d86f2b8a-7fb0-4d6c-b188-b258fd1b22db","added_by":"auto","created_at":"2025-12-15 06:41:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":620156,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMFP preserves cardiomyocyte viability by attenuating oxidative stress–induced apoptosis following myocardial infarction.\u003c/strong\u003e (A) Representative TUNEL-stained sections of infarct border zones from sham, MI, and MFP-treated MI hearts. (B) Quantification of TUNEL-positive nuclei showing significantly reduced apoptosis with MFP treatment. (C) Western blot analysis of apoptosis-related proteins in H₂O₂-exposed H9c2 cells, demonstrating MFP-mediated upregulation of Bcl-2 and suppression of Bax and cleaved caspase-3. (D) Flow cytometric Annexin V/propidium iodide analysis of H9c2 cells exposed to oxidative stress (H₂O₂) with or without MFP pre-treatment. (E) Quantitative analysis of apoptotic cell percentages from flow cytometry. Data indicate that MFP markedly mitigates oxidative stress–induced apoptosis both in vivo and in vitro, supporting its cardioprotective role in post-MI myocardial preservation.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8334699/v1/813471f79eff81020628c553.png"},{"id":98432386,"identity":"77284d81-12ae-4fcb-85b6-21ce43a219b6","added_by":"auto","created_at":"2025-12-17 16:49:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":311059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMFP mitigates oxidative stress and preserves mitochondrial integrity in myocardial infarction.\u003c/strong\u003e (A) Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in myocardial tissue from each treatment group (n = 5 per group). MFP restored SOD activity and reduced MDA accumulation compared with untreated MI. (B) Representative immunofluorescence images of MitoSOX Red staining in H9c2 cardiomyoblasts exposed to H₂O₂ with or without MFP pretreatment, showing reduced mitochondrial superoxide production with MFP. (C,D) Mitochondrial membrane potential (ΔΨm) assessment by JC-1 staining and flow cytometric quantification. H₂O₂ increased the green/red fluorescence ratio, indicating ΔΨm loss, whereas MFP preserved membrane potential. Data are mean ± SEM. *P \u0026lt; 0.05 versus indicated groups.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8334699/v1/1dd806dd5f1f211c86e2df56.png"},{"id":98195820,"identity":"9f390840-471b-4cee-ae08-837eacbde5e4","added_by":"auto","created_at":"2025-12-15 06:41:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":748228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMFP promotes robust angiogenesis and coronary microvascular regeneration post–myocardial infarction. \u003c/strong\u003eRepresentative immunohistochemical images of myocardial cryosections stained for the endothelial marker CD31 and angiogenic mediator VEGFR in sham-operated, MI, and MFP-treated MI groups. MFP markedly increased CD31-positive capillary density and VEGFR expression within the peri-infarct myocardium, indicating enhanced endothelial proliferation and activation of pro-angiogenic signaling. Quantification of staining intensity and capillary density is shown (mean ± SEM; n = 5 per group). Statistical significance: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8334699/v1/b2ba4bb355a7bae12c791521.png"},{"id":98195822,"identity":"27dcbc35-7094-4006-b3f0-2ebc4748cd11","added_by":"auto","created_at":"2025-12-15 06:41:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":787386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMFP suppresses post-infarction inflammation and drives reparative macrophage polarization. \u003c/strong\u003e(A) Representative myocardial cryosections from sham-operated, MI, and MFP-treated MI hearts stained for total macrophages (CD68), pro-inflammatory M1 macrophages (CD86), and reparative M2 macrophages (CD206). MFP markedly reduced CD68⁺ and CD86⁺ macrophage infiltration while increasing CD206⁺ macrophages in the peri-infarct region. (B) Flow cytometric analysis of Raw 264.7 macrophages demonstrating increased surface expression of CD206 and CD11b after 24 h MFP treatment, consistent with M2 polarization. (C) qRT-PCR analysis of PMA-stimulated THP-1 cells showing upregulation of M2-associated markers (CD163, IL-6, IL-10) and suppression of pro-inflammatory IL-1β following MFP exposure. Data are presented as mean ± SEM; n = 5 per group. Statistical significance: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8334699/v1/3e8cfa356aff836423a98f56.png"},{"id":98195853,"identity":"5322c5ee-d931-4f25-a3bc-fc5040606606","added_by":"auto","created_at":"2025-12-15 06:41:06","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":398871,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMFP reprograms oxidative stress–induced transcriptomic networks toward cardioprotection and immune resolution. \u003c/strong\u003e(A) Volcano plot of differentially expressed genes in H9c2 cardiomyocytes exposed to oxidative stress (600 μM H₂O₂) with or without MFP, showing 361 upregulated and 499 downregulated genes (|log₂FC| \u0026gt; 0.58, adjusted p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e(B) Ingenuity Pathway Analysis (IPA) “Tox Function” predictions indicating marked suppression of transcriptional signatures linked to pathological cardiac remodeling, including ventricular dilation, systolic dysfunction, and fibrosis. (C) Top positively and negatively enriched canonical pathways (z-score ≥ ±2, p ≤ 0.05) demonstrating modulation of cytokine signaling, mitochondrial homeostasis, and pro-survival cascades. (D) IPA-derived regulatory network highlighting MFP-induced upregulation of genes associated with M2 macrophage polarization (e.g., CD163, IL10, MRC1), consistent with a reparative immune phenotype.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8334699/v1/59de5b34745c6bd862075768.png"},{"id":98195842,"identity":"f69c707e-f09d-4848-a96a-eb5e43482752","added_by":"auto","created_at":"2025-12-15 06:41:06","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1206422,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThree-phase cardioprotective actions of MFP following acute myocardial infarction. \u003c/strong\u003eSchematic representation \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003cbr\u003e\n of the therapeutic effects of the multifunctional peptide (MFP) across the sequential phases of post–acute myocardial infarction (AMI) repair. During the inflammatory phase (days 1–4), MFP attenuates oxidative stress and suppresses inflammatory responses through reactive oxygen species scavenging. In the proliferative phase (days 4–14), MFP enhances endothelial cell activity and promotes angiogenesis to support tissue regeneration. In the maturation phase (beyond day 14), MFP reduces extracellular matrix deposition and facilitates reparative M2 macrophage polarization, thereby limiting fibrotic scar formation and improving myocardial remodeling.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8334699/v1/2b5a0a016fd81db472184259.png"},{"id":99799157,"identity":"e4c8404e-b08d-4dce-9e1f-121170abec4f","added_by":"auto","created_at":"2026-01-08 13:49:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9486994,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8334699/v1/361fbed7-5e5f-4702-a625-980e6caa97af.pdf"},{"id":98195848,"identity":"cb1c2698-71d5-4ece-bfdf-3471c1bc01c6","added_by":"auto","created_at":"2025-12-15 06:41:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":457333,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8334699/v1/7e7d6c8aba3ebbe7d5d3aa1b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Multifunctional Therapeutic Peptide Attenuates Post-Myocardial Infarction Remodeling Through Antioxidant, Pro-Angiogenic, and Immunomodulatory Mechanisms","fulltext":[{"header":"Background","content":"\u003cp\u003eMyocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide, primarily resulting from the abrupt occlusion of coronary arteries due to atherosclerotic plaque rupture and subsequent thrombus formation[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This ischemic event leads to an immediate and profound reduction in oxygen and nutrient supply to the myocardium, triggering a cascade of pathological processes that extend far beyond the initial vascular insult[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The pathological progression following myocardial injury encompasses myocardial ischemia, cardiomyocyte apoptosis, fibrosis, and exacerbation of the inflammatory response[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], collectively contributing to ventricular remodeling and structural and functional abnormalities and loss of functional cardiomyocytes within the heart[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], leading to more than 50% five-year mortality rate after MI[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAt the cellular level, ischemia instigates mitochondrial dysfunction and excessive production of reactive oxygen species (ROS), creating a highly oxidative environment that exacerbates cellular injury[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Oxidative stress, in turn, disrupts calcium homeostasis, damages cellular membranes, and activates intrinsic apoptotic pathways, leading to widespread cardiomyocyte death through both apoptosis and necrosis[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The resulting loss of functional myocardium impairs contractile performance and initiates the remodeling of the left ventricle.\u003c/p\u003e \u003cp\u003eThis initial injury is further complicated by a robust inflammatory response[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], characterized by the recruitment and activation of neutrophils, macrophages, and other immune cells[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While inflammation is essential for debris clearance and initiation of repair, excessive or prolonged inflammatory activity contributes to further tissue damage, promotes fibrotic scar formation, and impairs regenerative potential[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The transition from inflammation to resolution and repair is often dysregulated in MI, resulting in adverse ventricular remodeling marked by myocardial stiffening, thinning of the ventricular wall, and progressive heart failure. Consequently, the five-year mortality rate following MI remains unacceptably high, exceeding 50% in many populations.\u003c/p\u003e \u003cp\u003eGiven this pathophysiological complexity, current therapeutic strategies have shifted toward not only limiting acute injury but also promoting effective myocardial repair. Immunomodulatory interventions aimed at curbing excessive inflammation have demonstrated potential in halting ongoing damage[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]; however, they fall short in restoring contractile function or reversing structural deficits. In this context, therapeutic angiogenesis has emerged as a promising adjunctive strategy[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. By stimulating the formation of new blood vessels within the ischemic and peri-infarct regions, angiogenesis enhances perfusion, supports the metabolic demands of surviving cardiomyocytes, and facilitates functional recovery of the damaged myocardium[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA comprehensive approach targeting oxidative stress, modulating inflammation, and promoting angiogenesis may offer synergistic benefits in attenuating post-MI remodeling and improving long-term cardiac outcomes. Haptoglobin (Hp), an acute phase protein, has the potential to be a therapeutic protein for MI due to its antioxidant[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], anti-inflammatory[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], immune-regulatory[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and angiogenic properties. Hp's ability to scavenge free hemoglobin, limit ROS-mediated damage, modulate macrophage activity, and support neovascularization in ischemic tissue suggests it could be beneficial in treating MI[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. We identified and cloned the Hp alpha-1 (Hp α1) chain as a therapeutic fragment, with the goal of developing a multifunctional functional peptide (MFP) capable of recapitulating the protective effects of full-length Hp in a more targeted and controlled manner[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Given its favorable biological properties and potential for synthetic production and modification, the MFP\u0026ndash;derived peptide represents a novel therapeutic avenue for the treatment of MI, with the potential to simultaneously limit injury, modulate inflammation, and promote regeneration.\u003c/p\u003e \u003cp\u003eThis study explores the cardio protective potential of MFP in the context of MI, providing new insights into multifunctional protein-based strategies contribute to myocardial regeneration and functional restoration.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eHuman umbilical vein endothelial cells (HUVECs), H9c2 cardiomyoblast cells, and RAW264.7 murine macrophage cells were obtained from the Bioresource Collection and Research Center, Taiwan. HUVECs and H9c2 cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) (Gibco, USA, Cat# 11995040), while RAW264.7 cells were cultured in RPMI 1640 medium (Gibco, USA, Cat# 11875085). All media were supplemented with 10% fetal bovine serum (FBS) (Gibco, USA, Cat# 26140079) and 1% antibiotic-antimycotic solution (Gibco, USA, Cat# 15240062). Cells were incubated at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂ and 95% air.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMFP cloning, expression, purification and optimal\u003c/h3\u003e\n\u003cp\u003eMFP, modified from Hp α1 chain, was subcloned into a bacterial vector pET-30a(+) and this plasmid was transformed into \u003cem\u003eE. coli\u003c/em\u003e strain BL21(DE3) competent cells. The His-tagged MFP was expressed by an \u003cem\u003eE. coli\u003c/em\u003e expression system and purified by using immobilized metal affinity chromatography (MAM-50 His-NTA resin, EBL Biotechnology). Phosphate-buffered saline (PBS, pH 7.4) with 20 mM imidazole was used as binding buffer in this system, unbound proteins was washed out from the column by using PBS (pH 7.4) with 50 mM imidazole, and MFP was eluted by applying 50\u0026ndash;500 mM imidazole gradient. The eluate fractions were resolved on 12% SDS-PAGE, the fractions including MFP were combined, concentrated, and dialyzed into 1x PBS (pH 7.4). Endotoxin was removed by using Pierce High-Capacity Endotoxin Removal Resin (Thermo scientific).\u003c/p\u003e \u003cp\u003eIn conclusion, more than 10 mg of \u0026gt;\u0026thinsp;95% pure proteins were obtained per liter of \u003cem\u003eE. coli\u003c/em\u003e culture. MFP will be cloned and expressed by using an Escherichia coli (E. coli) expression system. The protein will be following purified by immobilized affinity chromatography.\u003c/p\u003e\n\u003ch3\u003eAntioxidant Activity Assay\u003c/h3\u003e\n\u003cp\u003eThe antioxidant capacity of test compounds was evaluated using a thiobarbituric acid-reactive substances (TBARS) assay, which quantifies lipid peroxidation products. Cu\u0026sup2;⁺ (copper sulfate) was used as an inducer of oxidative stress. Test substances included haptoglobin 1\u0026ndash;1 (Hp 1\u0026ndash;1) (Sigma-Aldrich), lysozyme, MFP, and probucol, which were each assessed across a range of concentrations (1.25 to 40 \u0026micro;M) using a 1:2 serial dilution scheme. In a typical assay, 4 \u0026micro;M CuSO4 (Sigma-Aldrich) and 40 mg of LDL were incubated with tested proteins.\u003c/p\u003e \u003cp\u003eFollowing incubation with Cu\u0026sup2;⁺ in the presence or absence of test proteins, the extent of lipid peroxidation was determined by measuring the formation of malondialdehyde (MDA)-TBA adducts spectrophotometrically. Antioxidant activity was quantified by the degree of inhibition of MDA formation compared to untreated controls.\u003c/p\u003e\n\u003ch3\u003eCell Proliferation Assay\u003c/h3\u003e\n\u003cp\u003eThe effect of MFP on endothelial cell viability and proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Human umbilical vein endothelial cells (HUVECs) were seeded at a density of 5,000 cells per well in 96-well plates (Falcon, Becton Dickinson, NJ) and incubated for 24 hours to allow cell attachment. Cells were then treated with MFP in conditioned media for 72 hours. After treatment, cells were washed with phosphate-buffered saline (PBS), and MTT solution (0.3 \u0026micro;g/\u0026micro;L in PBS; Sigma-Aldrich) was added to each well.\u003c/p\u003e \u003cp\u003ePlates were incubated at 37\u0026deg;C for 4 hours to allow the formation of formazan crystals. Subsequently, 100 \u0026micro;L of dimethyl sulfoxide (DMSO) was added to dissolve the crystals, and absorbance was measured at 565 nm using a microplate reader. Each experimental condition was performed in sextuplicate (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e\n\u003ch3\u003eIn Vitro Wound Healing Assay (Scratch)\u003c/h3\u003e\n\u003cp\u003eAn in vitro wound healing assay was performed to evaluate the effect of MFP on cell migration and wound closure under oxidative stress conditions. HUVECs were seeded into 6-well plates and cultured until reaching 95% confluence. A linear scratch was created across the cell monolayer using a sterile 200 \u0026micro;L pipette tip, followed by gentle washing with phosphate-buffered saline (PBS) to remove detached cells.\u003c/p\u003e \u003cp\u003eTo mimic oxidative injury, cells were treated with free hemoglobin (10 \u0026micro;M) as a source of reactive oxygen species. MFP was then applied at defined concentrations to assess its therapeutic effect on wound healing. Control and treatment groups were maintained in serum-free medium during the assay to exclude proliferation effects.\u003c/p\u003e \u003cp\u003ePhase-contrast images of the wound area were captured at 0 h and 8 h using an inverted microscope. Wound closure was quantified by measuring the distance between the wound edges using ImageJ software. The percentage of wound closure was calculated as:\u003c/p\u003e \u003cp\u003e[(initial wound width \u0026ndash; final wound width) / initial wound width] \u0026times; 100%.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn Vitro Angiogenesis Assay\u003c/h2\u003e \u003cp\u003eTo evaluate the pro-angiogenic effects of MFP, an in vitro capillary tube formation assay was performed using human umbilical vein endothelial cells (HUVECs) cultured on a growth factor-reduced extracellular matrix. Geltrex\u0026trade; basement membrane matrix (growth factor reduced; Invitrogen) was used to simulate the extracellular environment. Approximately 200 \u0026micro;L of matrix was added to 9 mm diameter cell culture insert wells with 0.45 \u0026micro;m pore size (Millipore), and allowed to polymerize at 37\u0026deg;C for 30 minutes.\u003c/p\u003e \u003cp\u003eHUVECs were resuspended in conditioned medium at a concentration of 1 \u0026times; 10⁴ cells/mL, and seeded onto the polymerized matrix at a density of 5 \u0026times; 10⁵ cells per well. Cells were incubated at 37\u0026deg;C for 16 hours to allow tube formation. After incubation, cells were stained with Calcein AM (Invitrogen) for 30 minutes to visualize live cells. Fluorescence images were captured using an Olympus BX51 fluorescence microscope.\u003c/p\u003e \u003cp\u003eThree random microscopic fields per well were selected, and capillary-like structures were quantified by measuring the total tube length using ImageJ software. The assay was performed in triplicate for each condition.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRat Model of Myocardial Infarction and Treatment Protocol\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eRat Model of Myocardial Infarction and Treatment Protocol\u003c/div\u003e \u003cp\u003eAdult male Sprague-Dawley (SD) rats (250\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g), obtained from LASCO (Taipei, Taiwan), were used to establish a chronic myocardial infarction (MI) model. MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery, as previously described. Successful LAD occlusion was confirmed intraoperatively by the presence of ST-segment elevation on electrocardiography (ECG) and visible myocardial surface cyanosis distal to the ligation site. Sham-operated animals underwent the same surgical procedure without LAD ligation.\u003c/p\u003e \u003cp\u003eFollowing surgery, LAD-ligated rats were randomly assigned to two groups: (1) saline control and (2) MFP treatment. In the MFP group, intramyocardial injections of MFP (1 \u0026micro;g/mL) were administered at five distinct sites within the peri-infarct (border) zone. Rats in the control and sham groups received an equivalent volume of sterile saline. All animals were monitored for four weeks post-MI.\u003c/p\u003e \u003cp\u003e All experimental procedures were conducted in accordance with the guidelines of the Animal Ethics Committee of Taipei Medical University and were approved prior to initiation of the study.\u003c/p\u003e\n\u003ch3\u003eEchocardiography\u003c/h3\u003e\n\u003cp\u003eTransthoracic echocardiography was performed to assess cardiac structure and function using a CX50 ultrasound system (Philips Ultrasound System, Andover, MA, USA). Rats were anesthetized with light isoflurane inhalation to maintain spontaneous breathing and minimize cardiac depression. The parasternal short-axis view of the left ventricle (LV) was obtained, and M-mode recordings were used to evaluate ventricular wall motion and chamber dimensions.\u003c/p\u003e \u003cp\u003eThe left ventricular internal diameter at end-diastole (LVIDd) and at end-systole (LVIDs) were measured. Left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were calculated using standard formulas:\u003c/p\u003e \u003cp\u003eLVEF (%) = [(LVIDd\u0026sup3; \u0026ndash; LVIDs\u0026sup3;) / LVIDd\u0026sup3;] \u0026times; 100\u003c/p\u003e \u003cp\u003eLVFS (%) = [(LVIDd \u0026ndash; LVIDs) / LVIDd] \u0026times; 100\u003c/p\u003e \u003cp\u003eAll measurements were performed in triplicate from three consecutive cardiac cycles and analyzed in a blinded manner to ensure objectivity.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHistological and TUNEL Analysis\u003c/h2\u003e \u003cp\u003eParaffin-embedded heart tissue sections (4 \u0026micro;m thick) were prepared for histological and apoptosis evaluation. Sections were deparaffinized in xylene and rehydrated through a graded ethanol series (100%, 90%, 70%) followed by a final rinse in distilled water.\u003c/p\u003e \u003cp\u003eFor general histology and fibrosis assessment, sections were stained with hematoxylin and eosin (H\u0026amp;E) or Masson\u0026rsquo;s Trichrome. In Masson\u0026rsquo;s Trichrome-stained sections, collagen fibers appeared blue, myocardial muscle fibers stained red, and nuclei stained blue-black, enabling clear delineation of fibrotic and viable tissue.\u003c/p\u003e \u003cp\u003eTo detect apoptotic cells, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using a commercial apoptosis detection kit (Roche, Branchburg, NJ, USA), following the manufacturer\u0026rsquo;s instructions. TUNEL-positive nuclei were visualized by fluorescence microscopy. The percentage of apoptotic cells was quantified by calculating the ratio of TUNEL-positive nuclei to total nuclei in randomly selected fields.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eImmunohistochemical analysis was performed on paraffin-embedded heart tissue sections to assess the expression of vascular, inflammatory, and immune cell markers. Sections (4 \u0026micro;m thick) were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval. Endogenous peroxidase activity was quenched using hydrogen peroxide. After rinsing, sections were blocked with 5% bovine serum albumin (BSA) to reduce nonspecific binding.\u003c/p\u003e \u003cp\u003eTissues were incubated overnight at 4\u0026deg;C with primary antibodies specific to the following markers: CD31 (1:2000; Abcam, Cat# ab182981), VEGFR (1:800; Abcam, Cat# ab9698), CD68 (1:100; Abcam, Cat# ab31630), CD163 (1:1000; Proteintech, Cat# 16646-1-AP), IL-6 (1:50; GeneTex, Cat# GTX17623), Control IgG (1:100; Abcam, Cat# ab37415)\u003c/p\u003e \u003cp\u003eFollowing primary antibody incubation, sections were treated with an HRP-conjugated secondary antibody and developed using 3,3'-diaminobenzidine (DAB) as the chromogen. Slides were then counterstained with hematoxylin, dehydrated, and mounted. Immunoreactive staining was visualized and evaluated under a light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometry Analysis of Mitochondrial Membrane Potential and Apoptosis\u003c/h2\u003e \u003cp\u003eMitochondrial membrane potential (ΔΨm) was assessed using the JC-1 assay kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s instructions. JC-1 dye selectively accumulates in mitochondria, forming red-fluorescent J-aggregates in cells with high ΔΨm, while remaining as green-fluorescent monomers in cells with depolarized (low ΔΨm) mitochondria. After experimental treatment, cells were incubated with JC-1 working solution at 37\u0026deg;C for 20 minutes in the dark, washed twice with dye buffer, and immediately analyzed by flow cytometry. The ratio of red (aggregates) to green (monomers) fluorescence intensity was used to quantify changes in ΔΨm.\u003c/p\u003e \u003cp\u003eIn parallel, apoptosis was assessed using Annexin V-FITC and propidium iodide (PI) staining (Invitrogen). Cells treated with hydrogen peroxide (H₂O₂), with or without MFP, were resuspended in binding buffer and incubated with Annexin V-FITC and PI for 15 minutes at room temperature in the dark. Samples were analyzed by flow cytometry to distinguish viable, early apoptotic, late apoptotic, and necrotic cell populations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Superoxide Detection\u003c/h2\u003e \u003cp\u003eMitochondrial superoxide production was assessed using MitoSOX\u0026trade; Red mitochondrial superoxide indicator (Invitrogen), following the manufacturer\u0026rsquo;s protocol. Cells were incubated with 2 \u0026micro;M MitoSOX Red reagent at 37\u0026deg;C for 30 minutes in the dark. After incubation, cells were washed with phosphate-buffered saline (PBS) to remove excess dye, and red fluorescence, indicative of mitochondrial superoxide accumulation, was visualized using a fluorescence microscope.\u003c/p\u003e \u003cp\u003eAs a positive control for mitochondrial dysfunction, cells were treated with 2 \u0026micro;M carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 30 minutes prior to MitoSOX staining to induce mitochondrial depolarization and elevated superoxide generation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Reverse Transcription PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells or tissues using TRIzol\u0026trade; reagent (Invitrogen), following the manufacturer\u0026rsquo;s instructions. Complementary DNA (cDNA) was synthesized from 1 \u0026micro;g of total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003cp\u003eQuantitative real-time PCR was performed using SYBR\u0026reg; Premix Ex Taq\u0026trade; II (Takara Bio) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Gene expression levels were normalized to the housekeeping gene GAPDH, and relative expression was calculated using the 2^\u0026ndash;ΔΔCt method.\u003c/p\u003e \u003cp\u003eThe primer sequences used were as follows: CD163: (Forward: 5\u0026prime;-GGCTAGACG AAGTCATCTGCAC-3\u0026prime;,\u0026emsp;Reverse: 5\u0026prime;-CTTCGTTGGTCAGCCTCAGAGA-3\u0026prime;), IL-6: (Forward: 5\u0026prime;-TACCACTTCACAAGTCGGAGGC-3\u0026prime;, \u0026emsp;Reverse: 5\u0026prime;-CTGCAAGTG CATCATCGTTGTTC-3\u0026prime;), IL-10: (Forward: 5\u0026prime;-CGGGAAGACAATAACTGCACC C-3\u0026prime;, Reverse: 5\u0026prime;-CGGTTAGCAGTATGTTGTCCAGC-3\u0026prime;), IL-1β: (Forward: 5\u0026prime;-TGGACCTTCCAGGATGAGGACA-3\u0026prime;,\u0026emsp;Reverse: 5\u0026prime;-GTTCATCTCGGAGCCTGT AGTG-3\u0026prime;), GAPDH: (Forward: 5\u0026prime;-TCACCACCATGGAGAAGGC-3\u0026prime;, Reverse: 5\u0026prime;-GCTAAGCAGTTGGTGGTGCA-3\u0026prime;)\u003c/p\u003e \u003cp\u003eAll reactions were run in triplicate, and melting curve analysis was conducted to ensure amplification specificity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blotting\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from cultured cells using RIPA lysis buffer supplemented with protease inhibitors (Thermo Scientific), followed by centrifugation at 14,000 \u0026times; g for 15 minutes at 4\u0026deg;C to remove debris. Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific).\u003c/p\u003e \u003cp\u003eEqual amounts of protein (20\u0026ndash;30 \u0026micro;g per sample) were separated via SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with 5% non-fat milk in TBS-T buffer (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature and incubated overnight at 4\u0026deg;C with the following primary antibodies: cleaved Caspase-3 (1:1000, Cell Signaling Technology, Cat# 9664), Bax (1:1000, Cell Signaling Technology, Cat# 2772), and Bcl-2 (1:1000, GeneTex, Cat# GTX100064). After washing, membranes were incubated with species-appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 1 hour at room temperature.\u003c/p\u003e \u003cp\u003eProtein bands were visualized using an enhanced chemiluminescence (ECL) detection substrate (Millipore) and imaged using a UVP BioImaging System. β-actin was used as a loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA Sequencing and Bioinformatics Network Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated and quantified, and high-quality RNA (1 \u0026micro;g) was used for library preparation using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA), following the manufacturer's protocol. Polyadenylated mRNA was enriched using oligo(dT)-conjugated magnetic beads and subsequently fragmented at elevated temperatures. First-strand cDNA synthesis was performed using random hexamer primers and reverse transcriptase, followed by second-strand synthesis to generate double-stranded cDNA.\u003c/p\u003e \u003cp\u003eAfter end repair, 3\u0026rsquo;-adenylation, and adaptor ligation, cDNA fragments were purified using the AMPure XP system (Beckman Coulter, Brea, CA, USA). Library quality and insert size distribution were assessed using the Agilent 2100 Bioanalyzer, and concentrations were validated using quantitative real-time PCR. Paired-end sequencing (150 bp reads) was carried out on an Illumina NovaSeq 6000 platform by Genomics, BioSci \u0026amp; Tech Co. (New Taipei City, Taiwan).\u003c/p\u003e \u003cp\u003eFor transcriptome analysis, raw sequencing reads were quality-filtered and aligned to the reference genome. Differentially expressed genes were identified and analyzed using Ingenuity Pathway Analysis (IPA, Qiagen, Redwood City, CA, USA) to elucidate regulatory networks, canonical signaling pathways, and functional gene associations in response to MFP treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Each experiment was independently repeated at least three times to ensure reproducibility. Statistical analyses were conducted using GraphPad Prism software (version 5.0; GraphPad Software, San Diego, CA, USA).\u003c/p\u003e \u003cp\u003eFor comparisons among multiple groups, one-way analysis of variance (ANOVA) was employed. Post hoc analyses were performed using Tukey\u0026rsquo;s multiple comparisons test for data with equal variances or the Dunnett\u0026rsquo;s T3 test when unequal variances were detected. All statistical tests were two-tailed, and a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMFP expression and purification\u003c/h2\u003e \u003cp\u003eTo produce recombinant MFP protein, the MFP coding sequence was subcloned into the pET-30a (+) bacterial expression vector, which contains a T7 promoter and His-tag sequences for affinity purification (Figure S1A). The resulting plasmid was transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) cells. Protein expression was induced, and His-tagged MFP was purified using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin under native conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe purification process involved binding in phosphate-buffered saline (PBS, pH 7.4) containing 20 mM imidazole, followed by a wash step with 50 mM imidazole to remove nonspecifically bound proteins. MFP was eluted using a linear imidazole gradient (50\u0026ndash;500 mM). Eluted fractions were analyzed by 12% SDS-PAGE stained with Coomassie blue (Figure S1B). MFP appeared as a prominent band at the expected molecular weight (~\u0026thinsp;25 kDa), with peak enrichment observed in fractions 6\u0026ndash;10. These fractions were pooled, concentrated, and dialyzed into PBS (pH 7.4).\u003c/p\u003e \u003cp\u003eFollowing endotoxin removal, the final preparation yielded more than 10 mg of \u0026gt;\u0026thinsp;95% pure MFP per liter of bacterial culture. These results confirm the robust expression and high-yield purification of recombinant MFP suitable for downstream functional and therapeutic studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMFP Exhibits Potent Antioxidant Activity Against Cu\u0026sup2;⁺-Induced Lipid Peroxidation\u003c/h2\u003e \u003cp\u003eOxidative stress is a well-established contributor to myocardial injury following acute myocardial infarction (AMI), largely through increased lipid peroxidation and inflammation[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. To assess the antioxidant capacity of MFP, a copper ion (Cu\u0026sup2;⁺)-induced low-density lipoprotein (LDL) peroxidation assay was employed. The extent of lipid peroxidation was measured via malondialdehyde (MDA) formation using a TBARS-based method.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, MFP demonstrated a strong, dose-dependent inhibition of MDA formation, indicating a robust antioxidant effect. Compared with known antioxidants, MFP was significantly more potent than both native Haptoglobin 1\u0026ndash;1 (Hp 1\u0026ndash;1) and probucol, an established lipid-lowering agent with antioxidant properties[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. While Hp 1\u0026ndash;1 and probucol exhibited moderate suppression of MDA production at higher concentrations, MFP nearly abolished MDA formation at concentrations as low as 10\u0026ndash;20 \u0026micro;M. In contrast, lysozyme, used as a negative control protein, showed no appreciable antioxidant activity across the tested concentration range.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings identify MFP as a highly effective antioxidant peptide, superior both endogenous Hp 1\u0026ndash;1 and pharmacological probucol in this in vitro model. Given the importance of oxidative injury in post-MI pathology, MFP may represent a promising candidate for therapeutic intervention targeting oxidative stress in AMI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMFP Enhances Endothelial Cell Proliferation, Migration, and Angiogenesis in Vitro\u003c/h2\u003e \u003cp\u003eEndothelial repair and angiogenesis are critical for tissue regeneration following acute myocardial infarction (AMI)[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Given the clinical relevance of vascular endothelial growth factor (VEGF) elevation post-AMI, we investigated whether MFP promotes endothelial cell function under oxidative stress conditions.\u003c/p\u003e \u003cp\u003eTo assess endothelial proliferation, human umbilical vein endothelial cells (HUVECs) were cultured in high glucose medium and treated with increasing concentrations of MFP. A dose-dependent increase in cell proliferation was observed, with a peak proliferative response (~\u0026thinsp;3-fold) at 0.125 mg/mL MFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Higher concentrations reduced this effect, suggesting an optimal therapeutic window.\u003c/p\u003e \u003cp\u003eNext, we evaluated endothelial migration using a scratch wound healing assay in the presence of hemoglobin (Hb), a known inducer of oxidative stress via reactive oxygen species (ROS) production[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Hb significantly impaired wound closure, whereas MFP supplementation reversed this inhibitory effect. By 8 hours, MFP-treated HUVECs achieving approximately 70% wound closure, compared with in the Hb-alone group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo assess angiogenic capacity, HUVECs were seeded on growth factor-depleted Matrigel to evaluate tube formation. MFP treatment robustly promoted angiogenesis, as evidenced by enhanced tube length and branch point formation. Notably, the angiogenic effect of MFP was comparable to that of VEGF, a key regulator of neovascularization [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e](Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eCollectively, these results demonstrate that MFP promotes endothelial proliferation, restores cell migration impaired by oxidative stress, and induces angiogenesis, underscoring its potential as a vascular regenerative therapy post-AMI.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMFP Promotes Endothelial Preserves Cardiac Function Following Myocardial Infarction\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEchocardiographic analysis was performed 14 days after MI induction to assess cardiac function[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. M-mode imaging revealed marked systolic dysfunction in MI animals, as shown by reduced left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), and increased left ventricular internal diameters during systole (LVIDs) and diastole (LVIDd) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Notably, MFP administration significantly preserved LVEF and LVFS and attenuated ventricular dilation compared to MI alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTogether, these findings suggest that MFP preserves cardiac structure and function in vivo after MI, highlighting its therapeutic potential for post-infarction remodeling and recovery.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMFP Attenuates Myocardial Fibrosis Post-Infarction\u003c/h2\u003e \u003cp\u003eMyocardial fibrosis is a hallmark of pathological cardiac remodeling following myocardial infarction (MI), contributing to impaired ventricular compliance and progression to heart failure[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. To assess whether MFP confers anti-fibrotic effects in vivo, histological and molecular analyses were conducted on cardiac tissues harvested four weeks after left anterior descending (LAD) artery ligation.\u003c/p\u003e \u003cp\u003eMasson's trichrome staining revealed extensive collagen deposition and wall thinning in the infarcted left ventricle of MI animals, indicative of substantial myocardial fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In contrast, MFP-treated animals exhibited markedly reduced fibrotic area and preserved myocardial structure. Quantitative analysis confirmed a significant reduction in interstitial collagen content in the MFP group compared to the untreated MI group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Hematoxylin and eosin (H\u0026amp;E) staining further demonstrated disorganized myocardial architecture and fibrotic infiltration in MI hearts, which were notably alleviated by MFP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunohistochemical staining for transforming growth factor-beta 1 (TGF-β1), a key mediator of fibrotic signaling, showed strong upregulation in the infarct region of MI hearts. MFP administration significantly suppressed TGF-β1 expression and reduced infarct size (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), supporting a direct anti-fibrotic mechanism.\u003c/p\u003e \u003cp\u003eTogether, these data indicate that MFP mitigates adverse fibrotic remodeling post-MI, potentially contributing to improved cardiac structure and function.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMFP Suppresses Post-Infarction Inflammatory Response\u003c/b\u003e \u003cb\u003evia\u003c/b\u003e \u003cb\u003eIL-6 Downregulation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExcessive inflammation following myocardial infarction (MI) contributes to adverse cardiac remodeling and impaired recovery[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. To investigate the anti-inflammatory effects of MFP, we evaluated interleukin-6 (IL-6) expression in infarcted cardiac tissue using immunohistochemistry.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, IL-6 expression was markedly elevated in the myocardium of MI animals compared to controls, consistent with an acute inflammatory response. Treatment with MFP significantly reduced IL-6 immunoreactivity in the infarct and peri-infarct regions, indicating attenuation of the inflammatory response. Quantification of IL-6-positive staining revealed a dramatic decrease in the MFP-treated group compared to the MI group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting that MFP mitigates inflammatory signaling after cardiac injury.\u003c/p\u003e \u003cp\u003eThese results highlight MFP\u0026rsquo;s immunomodulatory role in the post-MI setting, supporting its potential to limit inflammation-driven myocardial damage.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eMFP Attenuates Cardiomyocyte Apoptosis After Myocardial Infarction by Counteracting Oxidative Stress\u003c/h2\u003e \u003cp\u003eLoss of cardiomyocytes is a key driver of adverse left ventricular remodeling and subsequent cardiac dysfunction following myocardial infarction[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] (MI). Oxidative stress, particularly the overproduction of reactive oxygen species (ROS), plays a critical role in promoting cardiomyocyte apoptosis in the ischemic myocardium[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. To investigate the cardioprotective effect of MFP, we assessed both in vivo and in vitro markers of apoptosis.\u003c/p\u003e \u003cp\u003eTUNEL staining revealed a substantial increase in apoptotic cardiomyocytes in the infarct border zone of MI hearts compared to the sham-operated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;B), consistent with significant ischemia-induced cell death. Notably, MFP treatment significantly reduced the number of TUNEL-positive nuclei, indicating decreased apoptosis in the infarcted myocardium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the anti-apoptotic effect of MFP under oxidative stress, H9c2 cardiomyoblasts were exposed to hydrogen peroxide (H₂O₂, 600 \u0026micro;M) in vitro. Flow cytometric analysis with Annexin V/propidium iodide staining demonstrated a marked increase in apoptotic cells following H₂O₂ treatment (73.2% \u0026plusmn; 3%) compared to control (23.97%). Pre-treatment with MFP significantly attenuated H₂O₂-induced apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;E).\u003c/p\u003e \u003cp\u003eWestern blot analysis of apoptosis-related proteins corroborated these findings. H₂O₂ exposure led to increased expression of pro-apoptotic markers Bax and cleaved caspase-3, alongside decreased expression of the anti-apoptotic protein Bcl-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). MFP treatment reversed these trends, upregulating Bcl-2 while suppressing Bax and cleaved caspase-3 expression.\u003c/p\u003e \u003cp\u003eCollectively, these results suggest that MFP promotes cardiomyocyte survival following MI by counteracting oxidative stress-induced apoptosis and modulating key apoptotic signaling pathways.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eMFP Attenuates Oxidative Stress and Preserves Mitochondrial Function Following Myocardial Infarction\u003c/h2\u003e \u003cp\u003eOxidative stress is a major contributor to myocardial injury following infarction, promoting mitochondrial dysfunction and cardiomyocyte apoptosis[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To evaluate the antioxidative capacity of MFP in vivo, we quantified superoxide dismutase (SOD) activity and MDA levels in myocardial tissue[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Myocardial infarction significantly reduced SOD activity compared to the sham group; notably, MFP administration restored SOD levels toward baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In parallel, MDA\u0026mdash;an established marker of lipid peroxidation\u0026mdash;was markedly elevated in the MI group, whereas MFP treatment significantly decreased MDA levels, indicating reduced oxidative damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore MFP\u0026rsquo;s role in mitigating mitochondrial oxidative stress, we assessed mitochondrial superoxide generation using mitoSOX Red staining in H9c2 cardiomyoblasts. Exposure to hydrogen peroxide (H₂O₂) induced a substantial increase in mitochondrial ROS, evidenced by enhanced mitoSOX fluorescence. MFP pretreatment significantly suppressed H₂O₂-induced mitochondrial superoxide accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), suggesting a protective effect on mitochondrial redox balance.\u003c/p\u003e \u003cp\u003eGiven the importance of mitochondrial integrity in cell survival, we further evaluated mitochondrial membrane potential (ΔΨm) using JC-1 staining. H₂O₂ exposure led to a significant increase in the green/red fluorescence ratio, reflecting ΔΨm dissipation and mitochondrial depolarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;D). Remarkably, MFP treatment preserved ΔΨm by reducing the green/red fluorescence ratio, indicative of improved mitochondrial membrane stability.\u003c/p\u003e \u003cp\u003eThese findings demonstrate that MFP effectively counteracts oxidative stress and preserves mitochondrial function in cardiomyocytes, supporting its therapeutic potential in the context of myocardial infarction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eMFP Enhances Angiogenesis and Coronary Revascularization Following Myocardial Infarction\u003c/h2\u003e \u003cp\u003eEffective neovascularization is critical for myocardial repair and functional recovery following infarction[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. To determine whether MFP facilitates post-MI angiogenesis, we examined endothelial cell proliferation and vascular regeneration in infarcted myocardium via immunohistochemical analysis. CD31, an established endothelial marker, was used to assess capillary density in myocardial cryosections. Compared with the sham group, CD31 expression was markedly diminished in the MI group, indicating compromised microvascular integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Notably, treatment with MFP significantly restored CD31 expression, suggesting enhanced endothelial proliferation and neovascularization in the infarcted region.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, we assessed the expression of vascular endothelial growth factor receptor (VEGFR), a key mediator of angiogenic signaling. VEGFR levels were markedly elevated in MFP-treated hearts compared to both the MI and control groups, indicating potentiation of endogenous pro-angiogenic pathways.\u003c/p\u003e \u003cp\u003eCollectively, these findings demonstrate that MFP promotes angiogenesis in the infarcted myocardium, likely through upregulation of CD31 and VEGFR expression. This angiogenic activity may contribute to improved myocardial perfusion, reduced tissue injury, and enhanced cardiac repair post-infarction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eMFP Attenuates Inflammatory Cell Infiltration and Promotes Reparative Macrophage Polarization Following Myocardial Infarction\u003c/h2\u003e \u003cp\u003ePost-infarction inflammation is a key driver of adverse cardiac remodeling and heart failure progression[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. To evaluate the impact of MFP on inflammatory responses in the myocardium, immunohistochemical staining was performed to identify total macrophage infiltration (CD68), pro-inflammatory M1 macrophages (CD86), and anti-inflammatory M2 macrophages (CD206). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, myocardial tissues from the MI group displayed a substantial increase in CD68⁺ and CD86⁺ macrophages, indicative of pronounced inflammatory cell recruitment and M1 polarization. Strikingly, treatment with MFP significantly reduced both CD68⁺ and CD86⁺ macrophage infiltration, suggesting effective suppression of pro-inflammatory responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, CD206⁺ M2 macrophages were sparsely observed in infarcted myocardium, whereas MFP administration markedly increased CD206 expression, denoting a phenotypic switch towards reparative, anti-inflammatory macrophages. This shift in macrophage polarization toward the M2 lineage is associated with enhanced tissue repair, resolution of inflammation, and improved cardiac recovery.\u003c/p\u003e \u003cp\u003eTo further confirm the direct modulatory effect of MFP on macrophage polarization, Raw 264.7 macrophages were treated in vitro with MFP for 24 hours. Flow cytometric analysis revealed significantly elevated surface expression of CD206 and CD11b in MFP-treated cells, consistent with M2 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Gene expression profiling further supported this shift, showing increased mRNA levels of CD163, IL-6, and IL-10\u0026mdash;markers associated with anti-inflammatory M2 function\u0026mdash;while expression of the pro-inflammatory cytokine IL-1β was markedly downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eCollectively, these findings underscore the immunomodulatory potential of MFP in the post-MI setting. By reducing inflammatory macrophage infiltration and promoting M2 macrophage reprogramming, MFP not only mitigates inflammation but also fosters a reparative microenvironment conducive to myocardial healing. This dual anti-inflammatory and pro-reparative action positions MFP as a promising candidate for therapeutic intervention in post-infarction cardiac remodeling.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomic Profiling Reveals MFP Modulates Cardioprotective and Immunoregulatory Pathways in Oxidative-Stressed Cardiomyocytes\u003c/h2\u003e \u003cp\u003eTo gain mechanistic insight into the cardioprotective effects of MFP at the transcriptomic level, RNA-sequencing was performed on H9c2 cardiomyocytes exposed to oxidative stress (600 \u0026micro;M H₂O₂) with or without MFP treatment. A total of 860 differentially expressed genes (DEGs) were identified between the MFP- and PBS-treated groups, including 361 significantly upregulated and 499 downregulated genes (Log₂FC\u0026thinsp;\u0026gt;\u0026thinsp;0.58 or \u0026lt;\u0026thinsp;\u0026minus;\u0026thinsp;0.58, adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). These DEGs reflect MFP\u0026rsquo;s capacity to reprogram the stress-induced transcriptomic landscape of cardiomyocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIngenuity Pathway Analysis (IPA) of the DEGs revealed robust modulation of key canonical signaling pathways involved in inflammation, oxidative stress, cell survival, and repair. The top 10 significantly enriched canonical pathways in the MFP group included upstream regulators of cytokine signaling, mitochondrial homeostasis, and pro-survival cascades (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Importantly, IPA\u0026rsquo;s \"Tox Function\" analysis indicated marked suppression of molecular signatures linked to pathological cardiac remodeling. MFP treatment led to a substantial reduction in pathways associated with cardiac dysfunction, including ventricular dilation, systolic dysfunction, cardiomyocyte necrosis, and fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFurthermore, IPA identified a set of 12 MFP-regulated genes associated with M2 macrophage polarization, including CD163, IL10, and MRC1, providing molecular corroboration for the observed shift toward an anti-inflammatory reparative immune environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTogether, these transcriptomic findings suggest that MFP exerts its cardioprotective effects through a multifactorial mechanism involving modulation of redox-sensitive gene networks, suppression of deleterious cardiac stress pathways, and promotion of immunoresolving phenotypes. These data provide strong molecular evidence supporting MFP\u0026rsquo;s therapeutic potential in mitigating post-MI myocardial damage and maladaptive remodeling.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite significant advancements in reperfusion strategies and pharmacological therapies, myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide. While current treatment paradigms focus primarily on restoring coronary perfusion and mitigating acute complications, they often fail to address the complex cellular and molecular cascades triggered by ischemia-reperfusion injury\u0026mdash;namely oxidative stress, inflammation, endothelial dysfunction, and maladaptive ventricular remodeling[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Thus, there is an urgent need for novel therapies that not only limit initial injury but also promote myocardial repair and functional recovery.\u003c/p\u003e \u003cp\u003eIn this context, our study identifies MFP, a multifunctional therapeutic peptide, as a promising cardioprotective agent with significant translational relevance. We demonstrate that MFP exerts its effects through a multifaceted mechanism of action encompassing antioxidant activity, anti-apoptotic signaling, immunomodulation, and promotion of angiogenesis, ultimately culminating in improved cardiac structure and function post-MI.\u003c/p\u003e\n\u003ch3\u003eAnti-Remodeling and Anti-Fibrotic Effects\u003c/h3\u003e\n\u003cp\u003eLeft ventricular (LV) remodeling following MI is a pathophysiological continuum that encompasses infarct expansion, cardiomyocyte hypertrophy, and progressive interstitial fibrosis[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u0026mdash;all of which contribute to systolic dysfunction and heart failure. Our histological and molecular data confirm that MFP significantly attenuates myocardial interstitial fibrosis and collagen deposition. Mechanistically, this effect is associated with downregulation of TGF-β1, a key profibrotic cytokine known to drive fibroblast-to-myofibroblast differentiation and extracellular matrix accumulation. Additionally, MFP reduced the differentiates collagen expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), further supporting its role in preserving myocardial architecture and mitigating adverse LV remodeling.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eAnti-Apoptotic and Antioxidant Activity\u003c/h2\u003e \u003cp\u003eCardiomyocyte apoptosis is a hallmark of ischemia-reperfusion injury and contributes to progressive loss of viable myocardium post-infarction[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Our study shows that MFP treatment significantly reduced TUNEL-positive nuclei and suppressed pro-apoptotic markers Bax and cleaved caspase-3, while maintaining expression of the anti-apoptotic gene Bcl-2. These changes were accompanied by enhanced activation of the PI3K/Akt signaling pathway, which is well-documented for its cytoprotective role in the heart.\u003c/p\u003e \u003cp\u003eMFP also conferred robust antioxidant protection, as evidenced by decreased MDA levels and enhanced SOD activity[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Preservation of mitochondrial membrane potential in MFP-treated hearts suggests that mitochondrial integrity is maintained, likely contributing to improved cardiomyocyte survival. Transcriptomic analysis further reinforced these findings by revealing downregulation of oxidative stress-associated genes and suppression of cardiac dysfunction-related \u0026ldquo;Tox Functions,\u0026rdquo; including ventricular dilation, necrosis, and systolic failure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003ePro-Angiogenic and Endothelial Protective Effects\u003c/h2\u003e \u003cp\u003eNeovascularization within the infarct and peri-infarct regions is critical for restoring tissue perfusion and supporting myocardial regeneration[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. MFP promoted endothelial cell proliferation, migration, and tube formation in vitro, with efficacy comparable to that of VEGF. In vivo, this translated into increased capillary density and elevated VEGFR expression in the infarct border zone. These findings suggest that MFP enhances tissue revascularization and oxygen delivery, which are essential for optimal healing and functional recovery.\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eImmune Modulation and Macrophage Polarization\u003c/h2\u003e \u003cp\u003eUnresolved inflammation following MI exacerbates tissue damage and impairs repair[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. One of the most compelling aspects of MFP's action lies in its immunomodulatory capability. Our \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e data indicate that MFP shifts macrophage polarization from a pro-inflammatory M1 phenotype to a reparative M2 phenotype, as evidenced by reduced IL-6 expression, decreased M1 macrophage infiltration, and upregulation of M2 markers such as CD163, IL-10, and MRC1.\u003c/p\u003e \u003cp\u003eThis polarization shift likely involves multiple converging signaling pathways. For instance, activation of PI3K/Akt not only supports cardiomyocyte survival but also promotes M2 macrophage programming[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In parallel, modulation of PPAR, LXR/RXR, and Notch/TLR4 pathways may contribute to the observed immune reprogramming. Additionally, MFP may influence regulatory T-cell (Treg) activity and downstream cytokine cascades (e.g., IL-4, IL-10), which are known to support M2 polarization and angiogenesis via VEGF-A production. These findings highlight the intricate interplay between MFP and the immune system, underscoring its potential to modulate inflammation-resolving processes critical for cardiac repair.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eTranscriptomic Insights and Systems-Level Mechanisms\u003c/h2\u003e \u003cp\u003eRNA sequencing analysis identified 860 differentially expressed genes associated with MFP treatment, implicating critical pathways related to inflammation, oxidative injury, fibrosis, and immune regulation. Importantly, Ingenuity Pathway Analysis (IPA) predicted suppression of adverse cardiac phenotypes and activation of pathways involved in tissue repair. These systems-level insights provide a molecular foundation for the observed histological and functional improvements and underscore MFP's ability to orchestrate complex reparative responses in the infarcted myocardium.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eMFP as a Multi-Phase Modulator of Post-MI Cardiac Repair\u003c/h3\u003e\n\u003cp\u003eCardiac repair after myocardial infarction progresses through three interrelated phases[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u0026mdash;inflammatory, proliferative, and maturation\u0026mdash;each offering distinct therapeutic targets. MFP demonstrates a unique capacity to beneficially modulate all three stages. In the inflammatory phase (days 1\u0026ndash;4), MFP\u0026rsquo;s potent ROS-scavenging and anti-inflammatory properties attenuate oxidative stress and excessive immune activation, limiting collateral tissue damage and preserving viable myocardium. During the proliferative phase (days 4\u0026ndash;14), MFP enhances endothelial cell proliferation, accelerates endothelial wound closure, and promotes angiogenesis, improving perfusion to the peri-infarct zone while modulating macrophage polarization toward a reparative M2 phenotype. In the maturation phase (beyond day 14), MFP\u0026rsquo;s anti-fibrotic effects limit maladaptive extracellular matrix deposition, reduce scar stiffness, and preserve left ventricular compliance, thereby mitigating adverse remodeling.\u003c/p\u003e \u003cp\u003eBy simultaneously targeting oxidative stress, inflammation, neovascularization, and fibrosis in a temporally coordinated manner, MFP addresses the complex, sequential biology of post-MI repair with a single therapeutic agent\u0026mdash;an approach with clear translational promise for improving long-term cardiac outcomes.\u003c/p\u003e\n\u003ch3\u003eThree-phase cardioprotective actions of MFP after acute myocardial infarction\u003c/h3\u003e\n\u003cp\u003eThe therapeutic actions of the multifunctional peptide (MFP) across the three sequential phases of post\u0026ndash;acute myocardial infarction (AMI) repair (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e). In the inflammatory phase (days 1\u0026ndash;4), MFP mitigates oxidative stress and suppresses inflammation through reactive oxygen species scavenging. In the proliferative phase (days 4\u0026ndash;14), MFP supports pro-angiogenic endothelial cell activity and promotes tissue regeneration. In the maturation phase (beyond day 14), MFP exerts anti-fibrotic effects by reducing extracellular matrix deposition and enhancing reparative M2 macrophage polarization, thereby attenuating scar formation and improving myocardial remodeling[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom a therapeutic perspective, MFP offers several advantages: high-yield production, biological stability, and multimodal efficacy. Unlike conventional agents that typically target singular pathological mechanisms, MFP\u0026rsquo;s broad-spectrum activity addresses the multifactorial nature of MI pathology, potentially filling a major gap in the current treatment paradigm. The observed improvements in cardiac function, structure, and cellular viability post-MI suggest that MFP could serve as a first-in-class biologic for cardiac regeneration.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study provides compelling preclinical evidence that MFP is a potent and multifunctional therapeutic candidate capable of attenuating ischemic injury and enhancing myocardial repair following MI. Through coordinated modulation of fibrosis, apoptosis, oxidative stress, angiogenesis, and immune responses, MFP offers a promising avenue for regenerative cardiology. Future studies will be required to evaluate its long-term safety, pharmacokinetics, and therapeutic efficacy in large animal models and clinical trials. Nevertheless, these findings establish a strong foundation for the translational development of MFP and its potential to significantly impact the management of ischemic heart disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Myocardial infarction\u003c/p\u003e\n\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp; Reactive oxygen species\u003cbr\u003e\u0026nbsp;MFP \u0026nbsp; \u0026nbsp; \u0026nbsp; Multifunctional functional peptide\u003cbr\u003e\u0026nbsp;HUVECs \u0026nbsp; \u0026nbsp;Human umbilical vein endothelial cells\u003cbr\u003e\u0026nbsp;LAD \u0026nbsp; \u0026nbsp; \u0026nbsp; Left anterior descending\u003c/p\u003e\n\u003cp\u003eLV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Left ventricular\u003c/p\u003e\n\u003cp\u003eLVIDd \u0026nbsp; \u0026nbsp; Left ventricular internal diameter at end-diastole\u003c/p\u003e\n\u003cp\u003eLVIDs \u0026nbsp; \u0026nbsp; Left ventricular internal diameter at end-systole\u003c/p\u003e\n\u003cp\u003eLVEF \u0026nbsp; \u0026nbsp; \u0026nbsp;Left ventricular ejection fraction\u003c/p\u003e\n\u003cp\u003eLVFS \u0026nbsp; \u0026nbsp; \u0026nbsp;Left ventricular fractional shortening\u003c/p\u003e\n\u003cp\u003eANOVA \u0026nbsp; \u0026nbsp;One-way analysis of variance\u003c/p\u003e\n\u003cp\u003eMDA \u0026nbsp; \u0026nbsp; \u0026nbsp;Malondialdehyde\u003c/p\u003e\n\u003cp\u003eSOD \u0026nbsp; \u0026nbsp; \u0026nbsp;Superoxide dismutase\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at\u0026nbsp;https://doi.\u003c/p\u003e\n\u003cp\u003eorg/10.1186/sXXXX-XXX-XXXXX-X..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the technical support provided by the Core Facility Center and Laboratory Animal Center of Taipei Medical University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHYL, HYC, conducted experiments and acquired the data. WCC data statistic. CWC, CYC, and CP MFP purification. CHH MFP large scale production. FLM, KW, CHC, TRK and ATHW experiment design and paper write consult. CYH and CMS animal experiment and echocardiograms obtained. CCS and TMC wrote the manuscript and provided funding. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the National Science and Technology Council (NSTC), Taiwan (Grant No. NSTC 114-2314-B-038-091-MY3 to Tsai-Mu Cheng; NSTC 113-2314-B-038-010 and NSTC114-2314-B-038-158 to Alexander T.H. Wu; NSTC 113-2314-B-038-008 and NSTC 114-2314-B-038-156 to Chun-Ming Shih). Taipei Medical University, and the Higher Education Sprout Project by the Ministry of Education (DP2-TMU-114-O-01), Taiwan. Taipei Medical University-National Defense Medical Center Joint Research Program (TMU-NDMC-11304)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis published article and its supplementary information files include all data generated or analyzed during this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University and were approved prior to initiation of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTownsend N, Wilson L, Bhatnagar P, Wickramasinghe K, Rayner M, Nichols M. 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Front Immunol. 2019;10:300.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Multifunctional peptide, myocardial infarction, oxidative stress, macrophage polarization, angiogenesis, cardiac remodeling, immunomodulation","lastPublishedDoi":"10.21203/rs.3.rs-8334699/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8334699/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eMyocardial infarction (MI) triggers excessive oxidative stress, inflammatory activation, and maladaptive fibrosis, leading to adverse ventricular remodeling and heart failure. Therapeutic strategies capable of simultaneously modulating oxidative, inflammatory, and angiogenic pathways remain limited. This study investigates the cardioprotective effects of a novel Multifunctional Peptide (MFP) engineered to possess antioxidant, anti-inflammatory, pro-angiogenic, and endothelial-regenerative activities in a rat model of MI.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eAdult male Sprague\u0026ndash;Dawley rats underwent left anterior descending (LAD) coronary artery ligation to induce MI, followed by intramyocardial administration of MFP into peri-infarct myocardium. Cardiac remodeling and function were assessed via echocardiography and histopathology at 4 weeks post-MI. Macrophage phenotypes, fibrosis, angiogenesis, and apoptosis were evaluated using immunohistochemistry. In vitro studies examined MFP-mediated cytoprotection in H9C2 cardiomyocytes under oxidative stress and its effects on macrophage polarization in RAW 264.7 cells. RNA-sequencing was performed to identify transcriptomic signatures regulated by MFP.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eMFP treatment significantly improved left ventricular systolic function and attenuated post-MI structural deterioration. Histological analyses showed reduced cardiomyocyte apoptosis, diminished interstitial fibrosis, and markedly increased capillary density. MFP decreased pro-inflammatory macrophage infiltration while promoting reparative M2 macrophage polarization in vivo. In vitro, MFP protected cardiomyocytes against oxidative damage and enhanced M2-polarizing signaling in macrophages. Transcriptomic profiling revealed downregulation of genes associated with necrosis, inflammation, and adverse remodeling, and upregulation of pathways related to tissue repair, angiogenesis, and immune modulation.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e \u003cp\u003eMFP confers robust cardioprotection after MI by coordinating the suppression of oxidative stress and inflammation with the enhancement of angiogenic and reparative pathways. These findings support MFP as a promising therapeutic candidate for limiting infarct injury and improving myocardial repair.\u003c/p\u003e","manuscriptTitle":"A Multifunctional Therapeutic Peptide Attenuates Post-Myocardial Infarction Remodeling Through Antioxidant, Pro-Angiogenic, and Immunomodulatory Mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 06:40:41","doi":"10.21203/rs.3.rs-8334699/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ab225063-193c-4a53-b1cd-8568fbca4dc4","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-08T07:25:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 06:40:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8334699","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8334699","identity":"rs-8334699","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}