Targeted delivery of USP10 via neutrophil membrane-coated biomimetic nanoparticle facilitates cardiac repair through AMPK-dependent anti-apoptotic and pro-angiogenic pathways

Abstract

Background

Extensive loss of cardiomyocytes plays a major role in heart failure development after myocardial infarction (MI). Existing pharmacological treatments targeting cardiomyocyte apoptosis have shown limited clinical efficacy, largely because the underlying molecular mechanisms are unclear and effective drug delivery systems are unavailable. Emerging evidence suggests a critical regulatory role of ubiquitin-specific peptidase 10 (USP10) in cardiomyocyte apoptosis through multiple pathways.

Methods

USP10 expression and its association with cardiomyocyte apoptosis were evaluated in a murine MI model. A biomimetic nanoparticle coated with neutrophil membranes and encapsulating a USP10 overexpression plasmid (NM-NP-USP10) was developed and characterized. Subsequent investigations evaluated its biodistribution, biosafety, therapeutic efficacy, and underlying mechanisms both in vivo and in vitro. The role of AMPK signaling was examined through the application of the pharmacological inhibitor Compound C.

Results

USP10 expression was markedly downregulated in the infarcted cardiac tissue and showed a positive association with cardiomyocyte apoptosis. NM-NP-USP10 preferentially accumulated in the ischemic myocardium and exhibited favorable biosafety profiles. Moreover, treatment with NM-NP-USP10 not only attenuated cardiomyocyte apoptosis but also promoted angiogenesis in vivo. Mechanistically, NM-NP-USP10 activated the AMPK/Akt/eNOS signaling pathway. In contrast, inhibition of AMPK substantially abrogated its anti-apoptotic and pro-angiogenic effects, indicating that AMPK signaling serves as a critical mediator of USP10-induced cardioprotection.

Conclusions

These findings identify USP10 as a protective regulator in MI and demonstrate that targeted delivery of USP10 via neutrophil membrane-coated biomimetic nanoparticles facilitates cardiac repair through AMPK-dependent anti-apoptotic and pro-angiogenic mechanisms, underscoring its potential as a therapeutic strategy for MI.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12967-026-08591-3.

Introduction

Heart failure (HF) secondary to myocardial infarction (MI) is a leading risk factor of mortality in individuals with cardiovascular disease worldwide [1]. MI interrupts the supply of oxygen and nutrients essential for cardiomyocyte metabolism, triggering apoptosis and subsequent impairment of myocardial function [2]. This damage contributes to both systolic and diastolic dysfunction, substantially decreasing patient survival [3]. Although developments in pharmacological therapies and percutaneous coronary intervention have reduced in-hospital mortality, a major concern is the low regenerative capacity of adult cardiomyocytes [4]. Consequently, an infarcted myocardial tissue is frequently substituted by a fibrotic scar tissue, contributing to the persistently high prevalence of post-MI HF [1]. Therefore, it is critical to elucidate the molecular pathways that mediate cardiomyocyte death after infarction and to develop novel strategies for myocardial preservation and repair.

Ubiquitin-specific peptidases (USPs), a subgroup of deubiquitinating enzymes, catalyze ubiquitin moiety removal from substrate proteins or polyubiquitin chains in the normal physiological state [5, 6]. This enzymatic reaction is critical for regulating protein stability and function. Increasing evidence indicates that USPs contribute to the pathogenesis of several diseases, including neurodegenerative diseases, malignancies, and immune dysregulation [7–9]. In particular, recent studies support the role of USP10 in modulating apoptosis across multiple disease models. For instance, in a study by Xu et al. [10], USP10 activity suppression promoted apoptosis in myeloma cells by accelerating the degradation of cyclin D3. However, the specific involvement of USP10 in MI and its mechanistic influence on cardiomyocyte apoptosis remain largely undefined.

Biomimetic nanoparticles (NPs) have emerged as a novel class of drug delivery vehicles with several advantages, including tunable composition and size and superior biocompatibility because of their membrane-derived coatings [11, 12]. Notably, following MI, circulating neutrophils are swiftly recruited to ischemia site [13]. This recruitment is primarily mediated by interactions between neutrophil surface glycoproteins and selectins expressed on damaged endothelial cells [14]. These insights suggest that NPs coated with neutrophil membranes could facilitate targeted therapeutic agent delivery to the infarcted myocardial tissue.

Here, we assessed the biological function of USP10 in the pathophysiology of MI. We first analyzed USP10 expression in the cardiac tissue from MI mouse models. By using gain- and loss-of-function approaches, we then explored how USP10 modulates apoptosis under ischemic and hypoxic conditions and the potential signaling pathways involved in this process. Additionally, we engineered a neutrophil membrane-coated NP (NM-NP-USP10) loaded with a USP10 overexpression plasmid and administered it to mice with MI to assess its therapeutic efficacy.

Materials and methods

Animals

The Medical Ethics Committee of the First Affiliated Hospital of Xiamen University approved all animal experiments, and the study protocol followed international ethical guidelines. A mouse MI model was established using a modified protocol based on previous studies [15]. Following anesthesia induction with 5% isoflurane (RWD Life Science, China), male C57BL/6 mice [age: 10–12 weeks] underwent tracheal intubation and connection to a small animal ventilator for maintaining anesthesia with 1% isoflurane. After placing the mice in a supine position, the thoracic area was shaved and disinfected with iodine tincture. A left thoracotomy procedure was initiated through the fourth intercostal space, and the heart was accessed after opening the pericardium. Subsequently, the left anterior descending (LAD) coronary artery was permanently ligated with an 8 − 0 silk suture. The same surgical procedures, except for LAD ligation, were performed for sham group mice. For USP10 inhibition, Wu-5 (5 mg/mL in PBS; 10 µL) was administered by intramyocardial injection into the infarct zone during LAD ligation. The dosage used in this study was selected based on previously published studies demonstrating effective USP10 inhibition by Wu-5 and was applied throughout the study [16]. At 2 h after MI induction, experimental mice were subjected to NM-NP-USP10 treatment by administering 100 µg of NM-NP-USP10 in 100 µL PBS through the tail vein. To conduct localization studies in vivo, Cy5-labeled NPs were delivered through tail vein administration, and the distribution of these NPs within the infarcted myocardium was examined by immunofluorescence assay on day 3 post-injection. For clarity, “USP10” refers to free USP10 plasmid; “NP-USP10” refers to PLGA nanoparticles loaded with USP10 plasmids without neutrophil membrane coating; “NM-NP” refers to empty neutrophil membrane-coated nanoparticles without plasmid loading; “NM-NP-NC” refers to neutrophil membrane-coated nanoparticles loaded with scrambled control plasmids; and “NM-NP-USP10” refers to neutrophil membrane-coated nanoparticles loaded with USP10 plasmids. These terms are used consistently throughout the manuscript. All animal experiments received approval from the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Xiamen University (Approval No. 2021KYEC(069)) and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

Following the successful establishment of the MI model, animals were randomly assigned to various experimental groups using a randomized allocation method. Investigators responsible for outcome assessment and data analysis were blinded to group assignments whenever feasible. Animals that died during the surgical model establishment were excluded prior to randomization; however, no animals meeting the experimental criteria were excluded from subsequent analyses. Baseline characteristics, including body weight and cardiac function parameters, were comparable across groups before treatment administration.

Cardiac function assessment

Cardiac function was assessed by the Vevo 2100 echocardiography system (VisualSonics, Canada). The left ventricular internal diameter at end-systole (LVIDs) and end-diastole (LVIDd) were estimated through M-mode imaging in the short-axis view. Fractional shortening (FS%) and ejection fraction (EF%) were computed as follows: (1) FS% = [(LVIDd − LVIDs) / LVIDd] × 100% and (2) EF% = [(LVIDd³ − LVIDs³) / LVIDd³] × 100%.

NM-NP-USP10 construction and characterization

To prepare PLGA NPs, 50 mg PLGA-COOH (MCE, China) was dissolved in dichloromethane (3 mL) and sonicated. A solution containing 900 µL of a USP10 overexpression plasmid (20 µg/mL; VectorBuilder, China) and 100 µL of 0.1% (w/v) sodium cholate in Milli-Q water was added to the PLGA solution; next, this mixture was sonicated at 40% amplitude for 20 s in an ice bath. After adding 1% (w/v) sodium cholate (8 mL), the mixture was again sonicated for 40 s. Subsequently, 0.3% (w/v) sodium cholate (12 mL) was added; this mixture was then incubated for 3 h at room temperature (RT). The plasmid-loaded PLGA NPs were centrifuged for 15 min at 8500 × g and subsequently collected.

Neutrophil membranes were isolated from peripheral blood neutrophils of mice. NM-NP-USP10 was constructed by a direct extrusion method [17]. Equal weights of neutrophil membranes and NPs (1:1 w/w) were combined and sonicated for 60 s. A mini extruder (Avanti Polar Lipids, USA) was employed for passing the mixture through a polycarbonate membrane (200 nm) for 20 cycles to yield NM-NP-USP10. The morphology, size, and stability of the NPs were assessed by transmission electron microscopy (TEM; JEOL Ltd., Japan) and dynamic light scattering (DLS; Malvern, UK).

The polydispersity index (PDI) of the nanoparticles was assessed using dynamic light scattering (DLS) analysis. The encapsulation efficiency (EE) and loading capacity (LC) of USP10 plasmids were quantified by measuring the concentration of free plasmids remaining in the supernatant following nanoparticle preparation. EE and LC were calculated using the following equations: EE (%) = (amount of encapsulated plasmid / total plasmid amount) × 100%; LC (%) = (amount of encapsulated plasmid / total nanoparticle weight) × 100%.

To assess plasmid stability, naked USP10 plasmids, NP-USP10, and NM-NP-USP10 were incubated with DNase I, followed by analysis via agarose gel electrophoresis. The in vitro release profile of USP10 plasmids was evaluated by incubating the nanoparticles in PBS at 37 °C with gentle agitation. At predetermined intervals, the released plasmids were collected and quantified, and the cumulative release percentage was determined.

Membrane protein retention was assessed by comparing the protein profiles of neutrophil membranes and NM-NP-USP10 through SDS-PAGE, followed by staining with Coomassie Brilliant Blue. To evaluate cellular uptake, nanoparticles were labeled with DiO and incubated with cardiomyocytes. Fluorescence images were captured using a fluorescence microscope, and intracellular fluorescence intensity was quantified using ImageJ software.

Cell culture

Human umbilical vein endothelial cells (HUVECs; American Type Culture Collection) were grown in Endothelial Cell Medium (Servicebio, China) containing 5% fetal bovine serum. For internalization studies, HUVECs (2 × 10³ cells/well) were seeded on 4-chamber slides and co-incubated with Dio-labeled NM-NP-USP10 at 37 °C under 5% CO₂ for 6 h. Following a washing step using PBS to remove unbound particles, internalization was visualized by confocal microscopy (Leica Biosystems, Germany) and quantified.

Immunofluorescence and TUNEL staining

Heart tissue sections were dewaxed, rehydrated, permeabilized using Triton X-100 (0.1%), and blocked with bovine serum albumin (3%) at RT. Subsequently, the sections were incubated at 4 °C overnight with primary antibodies (Abcam, dilution: 1:200): cardiac troponin I (cTnI), platelet endothelial cell adhesion molecule-1 (CD31), and alpha-smooth muscle actin (α-SMA). After washing, the sections were exposed to appropriate fluorescent secondary antibodies at RT for 2 h. DAPI was utilized for counterstaining the nuclei. A confocal microscope was used for fluorescence imaging. For the quantification of angiogenesis, CD31-positive and α-SMA-positive vessels within the infarct border zone were analyzed in immunofluorescence-stained heart sections. Three randomly selected fields from each section were imaged at consistent magnification, and the number of positive vessels was manually counted using ImageJ software. The mean value derived from these three fields was calculated and used to represent each individual mouse. Results are presented as the number of positive vessels per square millimeter (mm²).

Apoptotic cardiomyocytes were identified with the TUNEL assay kit (Servicebio) by following the instructions on the kit. On day 3 post-MI, frozen sections of mouse heart were obtained after embedding in an OCT compound (Sakura, USA). The sections were then fixed with 4% paraformaldehyde for 10 min at RT and subsequently incubated with the TUNEL reaction mixture (50 µL) at 37 °C for 60 min. After PBS washing, immunofluorescence staining for cTnI and nuclear DAPI staining were conducted as described above. A confocal microscope was used to acquire images.

In vivo biodistribution analysis

To assess the in vivo biodistribution and myocardial targeting efficacy of NM-NP-USP10, fluorescence imaging was conducted following nanoparticle administration. Fluorescence intensity in major organs—including the heart, liver, spleen, lung, and kidney—was quantitatively analyzed using ImageJ software. Subsequently, these organs were harvested for further biodistribution evaluation. Total DNA was extracted from each tissue, and the relative abundance of USP10 plasmids was quantified via quantitative PCR (qPCR). Plasmid levels in the various organs were normalized to tissue weight to facilitate comparative analysis.

Serum biochemical analysis

To assess the systemic biosafety of NM-NP-USP10, peripheral blood samples were collected from mice at the conclusion of the experiment and centrifuged to isolate serum. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine were quantified using commercial assay kits in accordance with the manufacturers’ instructions. Subsequently, hepatic and renal function parameters were analyzed to evaluate potential systemic toxicity related to nanoparticle administration.

Masson’s trichrome staining

The staining process was performed as instructed by the manufacturer. Tissue sections were successively stained with iron hematoxylin (5 min) and ponceau acid fuchsin (5 min), rinsed in distilled water, and treated with phosphomolybdic acid (5 min), aniline blue (5 min), and 1% acetic acid (1 min). Stained sections were dehydrated, permeabilized using xylene, and mounted with resinene.

Lactate dehydrogenase (LDH) activity measurement

LDH activity in peripheral blood was estimated using a commercial LDH assay kit (Beyotime, China). Blood samples were centrifuged at 500 × g for 10 min, and the supernatant was diluted (1:9 (v/v) with deionized water. Subsequently, each well of a 96-well plate was filled with the diluted sample (50 µL) and the LDH assay reagent (50 µL); this was followed by incubation of the plate at RT for 30 min. LDH activity was quantified with a Synergy HT microplate reader (BioTek, USA).

Quantitative reverse transcription-polymerase chain reaction (RT-qPCR)

Total RNA extraction, cDNA synthesis, and RT-qPCR were conducted as reported earlier [17]. The internal control gene was Gapdh. Supplementary Table 1 shows details of the primer sequences.

Western blotting (WB) assay

Proteins were extracted from mouse heart tissue and estimated with a bicinchoninic acid protein assay kit (Servicebio). WB assay was conducted following the established protocols [17]. The primary antibodies utilized were as follows: USP10, endothelial nitric oxide synthase (eNOS), phosphorylated AMPK (p-AMPK), and AMPK (all from Abcam, dilution: 1:500) and phosphorylated Akt (p-Akt; Cell Signaling Technology, 1:500).

ELISA

TNF-α, IL-1β, IL-6, and reactive oxygen species (ROS) concentrations in heart tissues were quantified with ELISA kits (Yobibio, China) as instructed by the manufacturer.

Wound-healing assay

HUVECs were seeded in 6-well plates and cultured until reaching approximately 90% confluence. A linear scratch was created using a sterile 200-µL pipette tip, and detached cells were removed by washing with PBS. Subsequently, the cells were cultured in serum-free medium containing the specified treatments. Images were acquired at 0 h and 24 h post-scratch using an inverted microscope. The wound closure area was quantified using ImageJ software, and cell migratory capacity was expressed as the percentage of wound closure.

Tube formation assay

Matrigel (Corning, USA) was applied to 96-well plates and allowed to polymerize at 37 °C for 30 min. Subsequently, HUVECs were seeded onto the Matrigel-coated wells at the designated density and cultured with the appropriate treatments. Following a 6-hour incubation period, capillary-like tube structures were imaged using an inverted microscope. Quantification of tube formation was conducted by measuring the total tube length and the number of branch points using ImageJ software.

Statistical analysis

Mean ± standard deviation (SD) was used for reporting data. The sample size (n) for each experiment, including the number of animals and independent biological replicates, is indicated in the corresponding figure legends. Unpaired Student’s t-test was utilized for comparing two groups. One-way ANOVA was performed for comparing three or more groups, followed by Bonferroni’s post hoc test. The Kaplan–Meier survival curve was constructed for survival analysis. No animals were censored during the survival analysis. Pearson’s correlation coefficient (r) and its 95% confidence intervals were utilized for assessing relationships between continuous variables. Statistical significance was defined as P < 0.05. Data analysis and figure preparation were conducted using GraphPad Prism version 9.0 (GraphPad Software, USA).

Results

USP10 expression is reduced in mice heart following MI

A mouse MI model was successfully established. On day 3 post-infarction, ELISA showed that inflammatory mediators, including IL-1β, IL-6, TNF-α, and ROS, were markedly elevated in the myocardial tissue of MI mice (Fig. 1A–D). Compared to controls, the MI group exhibited high serum levels of LDH, a biomarker for myocardial injury (Fig. 1E), confirming the successful induction of MI. According to immunofluorescence staining, TUNEL-positive cardiomyocyte proportion in the heart tissue was remarkably higher in the MI group than in the control group; this was accompanied by a decreased USP10 fluorescence intensity relative to that in the control group (Fig. 1F–I). Further investigation demonstrated a negative correlation of USP10 expression with the proapoptotic genes Casp3, Casp11, and Bax, but a positive correlation with the antiapoptotic gene Bcl2 (Fig. 1J–M).

Fig. 1.

USP10 is involved in cardiomyocyte apoptosis during MI. (A–D) IL-1β, IL-6, TNF-α, and ROS concentrations in the infarcted myocardial tissue were quantified by ELISA. (E) Serum LDH activity was measured on day 3 following MI induction. (F, G) TUNEL-positive cardiomyocytes in the infarct region were detected by immunofluorescence staining. Scale bar = 100 μm. (H, I) Immunofluorescence staining was also performed for determining USP10 expression in the infarcted area. Scale bar = 100 μm. (J–M) Relationship between USP10 expression and apoptosis-related genes, including Casp3, Casp11, Bax, and Bcl2, was analyzed. N = 4–6 mice per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated by using Student’s t-test in (A-E, G and I), Pearson’s correlation analysis in (J-M)

Inhibition of USP10 expression by Wu-5 exacerbates cardiomyocyte apoptosis following MI

To evaluate how USP10 influences cardiomyocyte apoptosis post-MI, we administered Wu-5, a selective USP10 inhibitor, through intracardiac injection at the time of MI induction (Fig. 2A). The Wu-5 treatment group exhibited markedly higher ROS levels in the cardiac tissue and serum LDH levels than the PBS group (Fig. 2B, C). Immunofluorescence staining results indicated that, compared to the PBS group, the Wu-5 treatment group showed a high proportion of TUNEL-positive cardiomyocytes in the infarcted region (Fig. 2D, E). RT-qPCR also showed that the Wu-5 treatment group had higher Casp3 and Bax expression levels and a lower Bcl2 expression level than the PBS group (Fig. 2F–H).

Fig. 2.

Wu-5 inhibits the activity of cardiomyocytes and induces their apoptosis. (A) Timeline of the in vivo experimental procedure. (B) ROS levels in the infarcted myocardial tissue were quantified by ELISA. (C) Serum LDH activity was measured on day 3 after MI induction. (D, E) Immunofluorescence staining was conducted for detecting TUNEL-positive cardiomyocytes in the infarct zone. Scale bar = 100 μm. (F–H) Casp3, Bax, and Bcl2 expression was assessed by RT-qPCR. N = 4–6 mice per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated by using one-way ANOVA followed by Tukey’s post-hoc test in (B, C and E-H)

Synthesis and characterization of NM-NP-USP10

The structure of NM-NP-USP10 is shown in Fig. 3A. TEM analysis confirmed NP morphology (Fig. 3B). DLS measurements demonstrated that coating PLGA NPs with a neutrophil membrane increased both the particle diameter and the magnitude of the negative zeta potential (Fig. 3C, D). In addition, both NP-USP10 and NM-NP-USP10 exhibited low polydispersity indices, indicating a uniform size distribution (Figure S1A). Analyses of encapsulation efficiency and loading capacity further validated the effective incorporation of USP10 plasmids into the nanoparticle delivery system (Figure S1B, C). Stability assays revealed that particle size remained consistent throughout incubation, while agarose gel electrophoresis confirmed that nanoparticle encapsulation protected USP10 plasmids from DNase I-mediated degradation (Fig. 3E and Figure S1D). Release kinetics analysis indicated a sustained release profile of USP10 plasmids from both NP-USP10 and NM-NP-USP10, with membrane-coated nanoparticles exhibiting a comparatively slower release rate (Figure S1E). Additionally, Coomassie Brilliant Blue staining demonstrated that NM-NP-USP10 preserved the characteristic membrane protein profile of neutrophil membranes, confirming the successful retention of biomimetic membrane components (Figure S1F). Cellular uptake assays revealed efficient internalization of DiO-labeled nanoparticles by cardiomyocytes (Figure S1G, H). Consistent with findings in HUVECs (Fig. 3F, G), NM-NP-USP10 showed enhanced cellular uptake relative to uncoated nanoparticles, further substantiating the effective delivery capability of the biomimetic nanoparticle platform.

Fig. 3.

Synthesis and characterization of NP NM-NP-USP10. (A) Schematic representation of the structure of NM-NP-USP10. (B) TEM was used to examine NP morphology. Scale bar = 100 nm. (C, D) DLS was performed for determining particle size distribution and zeta potential. (E) NP stability was evaluated in serum. (F, G) Immunofluorescence staining was conducted to confirm cellular internalization of NPs. Scale bar = 100 μm. N = 4 independent experiments per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated by using using Student’s t-test in (C, D and G)

NM-NP-USP10 preferentially accumulates in ischemic myocardium and demonstrates favorable biosafety

The distribution of NM-NP-USP10 within the heart was further validated in a mouse model of MI (Fig. 4A). RT-qPCR analysis showed that NM-NP-USP10 increased USP10 expression in infarcted myocardial tissue compared to NP-USP10, indicating efficient in vivo delivery of the therapeutic plasmid (Fig. 4B). Immunofluorescence analysis consistently showed significantly higher accumulation of NM-NP-USP10 in the infarct region compared to NP-USP10 (Fig. 4C, D). To evaluate biodistribution, USP10 plasmid levels were quantified in major organs. The results demonstrated a higher accumulation of NM-NP-USP10 in the heart relative to NP-USP10, with minimal detection in the liver, spleen, lung, and kidney (Fig. 4E), thereby confirming the myocardial-targeting capability of the neutrophil membrane-coated nanoparticles. Serum biochemical analyses showed no significant alterations in ALT, AST, urea, or creatinine levels following NM-NP-USP10 administration (Fig. 4F), indicating favorable systemic biosafety of the nanoparticle platform.

Fig. 4.

NM-NP-USP10 preferentially accumulates in the ischemic myocardium with favorable biosafety. (A) The in vivo experimental protocol timeline. (B) RT-qPCR was conducted for estimating Usp10 gene expression in the myocardial tissue. (C, D) Immunofluorescence analysis of NM-NP-USP10 distribution in the infarcted myocardium. Scale bar = 100 μm. (E) The biodistribution of USP10 plasmids in the heart, liver, spleen, lung, and kidney was quantified using RT-qPCR. (F) Serum biochemical analyses, including measurements of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine levels, were conducted to assess the systemic biosafety of NM-NP-USP10. N = 4 mice per group. Quantitative data are presented as mean ± SD. Statistical significance was determined using Student’s t-test for panels (B, D, E, and G)

NM-NP-USP10 enhances cardiac function and mitigates adverse cardiac remodeling following myocardial infarction

As additional controls, the NP-USP10 and NM-NP-NC formulations were evaluated in preliminary experiments. Neither formulation produced a significant improvement in cardiac function nor a reduction in infarct size compared to the MI group (Figure S2A–C), thereby underscoring the necessity of the complete NM-NP-USP10 formulation. In the murine MI model, treatment with NM-NP-USP10 significantly upregulated Usp10 expression in the infarcted myocardium, as demonstrated by RT-qPCR analysis (Figure S2D). Furthermore, myocardial ROS levels and serum LDH activity were substantially decreased after NM-NP-USP10 administration (Figure S2E and S2F), suggesting a mitigation of myocardial injury.

The therapeutic efficacy of NM-NP-USP10 was further evaluated in vivo. The 28-day survival rate of mice with MI was significantly enhanced following treatment with NM-NP-USP10 (Fig. 5A). Measurements of the heart weight-to-body weight ratio (HW/BW) and echocardiographic evaluations demonstrated that NM-NP-USP10 attenuated post-infarction cardiac dilation and ventricular remodeling (Fig. 5B–E). Correspondingly, the group treated with NM-NP-USP10 exhibited markedly increased LVEF and LVFS, indicating notable improvement in cardiac function (Fig. 5F, G). Masson’s trichrome staining further revealed a reduction in myocardial fibrosis and an increase in left ventricular wall thickness following NM-NP-USP10 administration (Fig. 5H–J). Consistent with these histological findings, RT-qPCR analysis indicated a significant downregulation of fibrosis-associated genes Col1a1 and Col3a1 in the infarcted myocardium (Fig. 5K, L). Collectively, these results suggest that NM-NP-USP10 effectively mitigates myocardial injury, enhances cardiac function, and reduces adverse cardiac remodeling subsequent to MI.

Fig. 5.

NM-NP-USP10 facilitates cardiac repair after myocardial injury. (A) Kaplan–Meier survival curve. (B) HW/BW ratio. (C–G) Cardiac function was evaluated by echocardiography, including determination of EF (%), LVIDs (mm), LVIDd (mm), and FS (%). (H–J) Ventricular wall thickness and fibrotic area were quantified by Masson’s trichrome staining. (K, L) Col1a1 and Col3a1 gene expression levels were quantified by RT-qPCR. Statistical comparisons were achieved with one-way ANOVA followed by Bonferroni’s post hoc test. N = 4–10 mice per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated by using one-way ANOVA followed by Tukey’s post-hoc test in (B, D-G, I, J, K, and L)

NM-NP-USP10 activates the AMPK/Akt/eNOS signaling pathway in an AMPK-dependent manner

Previous studies have indicated that USP10 may play a regulatory role in AMPK signaling. Western blot analysis demonstrated that NM-NP-USP10 significantly increased USP10 protein expression compared to the control groups (Fig. 6A and B). Moreover, NM-NP-USP10 substantially enhanced AMPK phosphorylation and upregulated the expression of downstream signaling molecules, including p-Akt and eNOS (Fig. 6C–F). Notably, treatment with the AMPK inhibitor Compound C markedly inhibited NM-NP-USP10-induced AMPK activation and reduced the upregulation of p-Akt and eNOS. These results suggest that the activation of the Akt/eNOS signaling pathway by NM-NP-USP10 is at least partially dependent on AMPK signaling.

Fig. 6.

NM-NP-USP10 activates the AMPK/eNOS signaling pathway. (A–F) USP10, AMPK, p-AMPK, p-Akt, and eNOS protein levels were determined by western blot analysis across the specified treatment groups, including NM-NP-USP10 and NM-NP-USP10 combined with the AMPK inhibitor Compound C. N = 4 mice per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated by using one-way ANOVA followed by Tukey’s post-hoc test in (B-F)

Activation of AMPK signaling mediates the anti-apoptotic effects of NM-NP-USP10

TUNEL staining demonstrated a marked decrease in the number of apoptotic cardiomyocytes within the infarct region subsequent to NM-NP-USP10 treatment (Fig. 7A and B). Notably, pharmacological inhibition of AMPK using Compound C significantly diminished this protective effect, leading to an elevated number of TUNEL-positive cardiomyocytes. Corroborating these findings, RT-qPCR analysis revealed that NM-NP-USP10 treatment significantly downregulated the expression of the pro-apoptotic genes Casp3 and Bax, while upregulating the anti-apoptotic gene Bcl2. These effects were partially reversed upon administration of Compound C (Fig. 7C–E).

Fig. 7.

NM-NP-USP10 inhibits cardiomyocyte apoptosis through AMPK signaling. (A, B) Apoptotic cardiomyocytes within the infarct region were identified using TUNEL staining. Scale bar = 100 μm. (C–E) The expression levels of Casp3, Bax, and Bcl2 were assessed via RT-qPCR. (F) Representative Western blot images of pro-caspase-3 and cleaved caspase-3. (G, H) Quantitative analysis of pro-caspase-3 and cleaved caspase-3 protein expression. N = 4–6 independent experiments per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post-hoc test in (B-E, G and H)

To further substantiate the anti-apoptotic mechanism at the protein level, western blot analysis was conducted to assess caspase-3 activation. Treatment with NM-NP-USP10 resulted in an increased expression of pro-caspase-3 and a significant reduction in cleaved caspase-3 levels, indicating suppression of caspase-3 activation. Importantly, inhibition of AMPK signaling by Compound C partially reversed these effects, leading to elevated cleaved caspase-3 expression. These results further corroborate that the activation of AMPK signaling mediates the anti-apoptotic effects of NM-NP-USP10 (Fig. 7F–H).

Activation of AMPK signaling mediates the pro-angiogenic effects of NM-NP-USP10

Given the established role of AMPK signaling in promoting angiogenesis [18, 19], we investigated whether NM-NP-USP10 enhances neovascularization in the infarcted heart. Immunofluorescence staining revealed that NM-NP-USP10 treatment significantly increased the density of α-SMA-positive arterioles and CD31-positive capillaries within the infarcted region. Quantitative analysis, conducted by counting positive vessels in randomly selected fields, demonstrated a pronounced enhancement of vascular density following NM-NP-USP10 administration. Notably, these effects were markedly diminished upon treatment with the AMPK inhibitor Compound C (Fig. 8A and B).

Fig. 8.

NM-NP-USP10 promotes angiogenesis through AMPK signaling. (A) Representative immunofluorescence images depicting α-SMA-positive vessels within the infarct region are presented alongside a quantitative analysis of arteriole density. (B) Representative immunofluorescence images of CD31-positive capillaries in the infarct region are shown with corresponding quantitative analysis of capillary density. Quantification of CD31-positive and α-SMA-positive vessel densities was conducted by counting positive vessels in three randomly selected fields per section, with the average value calculated for each mouse. Scale bar = 100 μm. (C, D) Representative images and quantitative analyses of endothelial cell migration, assessed via wound-healing assay, and endothelial tube formation are provided. Scale bar = 100 μm. (E–H) Expression levels of Vegf, Ang1, Del-1, and Fgf were measured using RT-qPCR. N = 4 independent experiments per group. Quantitative data are presented as mean ± SD. Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post-hoc test in (A-H)

To further assess the pro-angiogenic activity of NM-NP-USP10, endothelial cell migration and tube formation assays were conducted. NM-NP-USP10 significantly enhanced endothelial cell migratory capacity and facilitated the formation of capillary-like structures, whereas inhibition of AMPK signaling substantially abrogated these effects (Fig. 8C and D). Consistent with these observations, RT-qPCR analysis revealed that NM-NP-USP10 markedly upregulated the expression of several angiogenesis-related genes, including Vegf, Ang1, Del-1, and Fgf. Notably, treatment with Compound C partially reversed the induction of these pro-angiogenic genes (Fig. 8E–H). Collectively, these results suggest that NM-NP-USP10 promotes angiogenesis both in vivo and in vitro, and that activation of AMPK signaling is at least partially required for its pro-angiogenic effects.

Discussion

We analyzed the critical role of USP10 in the pathophysiological processes underlying MI. Based on the obtained insights, we developed a biomimetic NP system encapsulating a USP10 overexpression plasmid and evaluated its therapeutic efficacy in a murine model of MI. The main findings are as follows. First, USP10 expression was markedly reduced in the infarcted myocardial tissue and exhibited a positive correlation with cardiomyocyte apoptosis. Second, USP10 activity inhibition exacerbated cardiomyocyte apoptosis in the infarct zone. Third, neutrophil membrane-coated PLGA NPs exhibited significant accumulation in the infarcted region. Fourth, treatment with NM-NP-USP10 suppressed cardiomyocyte apoptosis by AMPK/eNOS signaling pathway activation, thereby enhancing angiogenesis in the infarct area. Fifth, NM-NP-USP10 administration improved cardiac function, reduced fibrosis, and facilitated myocardial repair after MI.

The inability of adult mammalian cardiomyocytes to regenerate represents a major limitation in the effective treatment of MI [20]. In response to the massive cardiomyocyte loss caused by MI, current therapeutic approaches primarily aim to supplement exogenous cardiomyocytes or preserve the viability of endogenous cells in the infarct region [21, 22]. Human pluripotent stem cell-derived cardiomyocytes (HPSC-CMs) can be transplanted into the infarcted myocardium. However, recent studies revealed that the survival rate of HPSC-CMs is considerably low under the ischemic and hypoxic conditions typically found in the infarcted heart [23]. Moreover, some studies suggest that HPSC-CM transplantation may increase the risk of malignant arrhythmias such as ventricular tachycardia [23]. In contrast, strategies aimed at preserving the viability of native cardiomyocytes are considered safer and more effective for MI therapy [24]. The inhibition of cardiomyocyte apoptosis during MI can mitigate adverse ventricular remodeling and delay the onset of HF [25]. According to recent studies, USP10 participates in regulating both cell cycle and apoptosis. Located on chromosome 16q24.1, Usp10 encodes a USP family member, which modulates target protein activation and stability through deubiquitination [26]. The present study confirmed that USP10 expression is remarkably downregulated during MI, and its inhibition accelerates cardiomyocyte death. These findings provide mechanistic insights and highlight USP10 as a potential target for MI therapy. Although Wu-5 was employed as a pharmacological inhibitor of USP10 in the present study, the possibility of off-target effects associated with small-molecule inhibitors cannot be entirely excluded. Consequently, future investigations would benefit from incorporating additional genetic approaches, such as siRNA- or shRNA-mediated knockdown, to further confirm the specificity of USP10-related mechanisms.

Promoting angiogenesis within the infarcted myocardium has been recognized as an effective therapeutic approach for MI. Li et al. demonstrated that microvesicle-derived miR-486-5p enhanced myocardial repair in a non-human primate MI model by promoting angiogenesis [27]. Our present study confirmed that USP10 overexpression in the infarcted myocardium activates the AMPK signaling pathway, which promotes angiogenic processes [18]. Accordingly, we assessed angiogenesis in the infarct zone 28 days after NM-NP-USP10 administration. The results showed a significant increase in α-SMA-positive small arteries and CD31-positive capillaries in the treated group. Furthermore, according to RT-qPCR analysis, angiogenesis-related genes showed elevated expression in NM-NP-USP10-treated hearts. These findings suggest that NM-NP-USP10 could facilitate myocardial repair through dual mechanisms: cardiomyocyte apoptosis inhibition and angiogenesis promotion.

Recent progress in NP-based drug delivery systems has substantially improved the precision and therapeutic efficacy of pharmacological interventions [28]. Surface modification of NPs has proven particularly effective in enhancing their ability to cross biological barriers and achieve organ-specific targeting [29]. Among various surface engineering approaches, targeting peptides have been widely employed; however, their susceptibility to proteolytic degradation and short half-life have limited their effectiveness in vivo. In contrast, biomimetic NPs coated with biological membranes offer improved stability and biocompatibility, representing an innovative and promising strategy to achieve targeted drug delivery [11]. Here, we engineered NPs coated with neutrophil membranes and found that this modification significantly enhanced their accumulation in the infarcted myocardial tissue and increased the therapeutic efficacy of USP10 overexpression in a murine MI model.

Despite the promising therapeutic effects demonstrated in the present study, several limitations should be acknowledged. First, although our data support the involvement of AMPK signaling in mediating the cardioprotective effects of NM-NP-USP10, the mechanistic conclusions are primarily based on pharmacological inhibition experiments. In addition, the functional role of USP10 was investigated using the small-molecule inhibitor Wu-5. Although Wu-5 has been previously reported as a USP10 inhibitor, potential off-target effects cannot be completely excluded. Therefore, the mechanistic findings should be interpreted with appropriate caution. Future studies employing genetic approaches, such as cardiomyocyte-specific USP10 knockdown or knockout models, as well as genetic modulation of AMPK signaling, will be valuable for further validating the USP10–AMPK signaling axis in myocardial infarction. Second, the present study primarily focused on the early to intermediate stages of post-MI repair. Accordingly, longer-term follow-up studies will be necessary to determine the durability of the therapeutic benefits and to further evaluate long-term safety. Finally, although preliminary biodistribution and biosafety assessments demonstrated favorable cardiac targeting and no apparent systemic toxicity, several challenges remain before clinical translation can be considered. These include large-scale manufacturing, batch-to-batch reproducibility, scalability, regulatory compliance, and validation in large-animal models. Addressing these issues will be essential for the future development and potential clinical application of the NM-NP-USP10 platform.

Conclusion

The present study demonstrates that USP10 expression is significantly downregulated during MI and is positively associated with cardiomyocyte apoptosis. Treatment with NM-NP-USP10 strengthened cardiac function and alleviated myocardial fibrosis. NM-NP-USP10 induced therapeutic effects by inhibiting cardiomyocyte death and enhancing vascular regeneration in the infarcted region, primarily by activating the AMPK signaling pathway.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

JZ, JX and WJ: designing study, conducting experiments, analyzing data, and writing manuscript. JH, XZ, ZZ, GC, HB and LL: performing experiments and editing manuscript. ZS: designing experiments, analyzing data, and editing manuscript. All authors read and approved the final manuscript.

Funding

This study was funded by the Natural Science Foundation of Fujian Province, China (2022J011373).

Data availability

The data supporting the results of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments received approval from the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Xiamen University (Approval No. 2021KYEC(069)).

Consent for publication

Not applicable.

Competing interests

None.