Mesenchymal stem cell-derived exosomal mir-21-5p inhibits YAP1 expression and improves outcomes in myocardial infarction

Abstract

Background

Myocardial infarction (MI) remains a significant global health concern, characterized by cardiomyocyte apoptosis and adverse ventricular remodeling. Nevertheless, the interplay between exosomal miR-21-5p and Yes-associated protein 1 (YAP1) in the context of MI remains unexplored.

Methods

Rat mesenchymal stem cells (MSCs) and H9c2 cardiomyocytes were cultured, characterized, and instrumental in our experiments. Exosomes were meticulously isolated, and their identity confirmed. The internalization of these exosomes by H9c2 cells was assessed, while RNA and protein expression were quantified using Quantitative Real-Time PCR and Western blot, respectively. MTT assay was implemented for cell viability, and apoptosis was evaluated via flow cytometric analysis. To elucidate gene interactions, we conducted microarray profiling of miRNA expression, dual luciferase reporter assays, and RNA Immunoprecipitation.

Results

MSC-derived exosomes exhibited a remarkable capacity to attenuate hypoxia-induced inflammation and apoptosis in H9c2 cells. Notably, these exosomes significantly upregulated miR-21-5p levels within H9c2 cells, and the abrogation of miR-21-5p function abated their protective effects. Through computational analysis, we unveiled a miR-21-5p binding site in the 3’UTR of YAP1, which directly inhibited YAP1 expression. Importantly, the inhibition of YAP1 effectively reinstated the protective effects of exosomes in cells with impaired exosomal miR-21-5p.

Conclusion

This study underscores the pivotal role played by MSC-derived exosomes in safeguarding against MI, primarily by mediating the transfer of miR-21-5p, which targets YAP1 signaling pathways.

Clinical trial number

N/A.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12872-024-04197-z.

Highlights

MSC-derived exosomes inhibit inflammation and apoptosis

Exosomes significantly increase miR-21-5p expression.

MiR-21-5p directly targets and inhibits YAP1.

Exosomes act through the miR-21-5p/YAP1 axis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12872-024-04197-z.

Introduction

Myocardial infarction (MI), stemming from coronary artery blockage, poses a major global health burden, triggering cardiomyocyte apoptosis and adverse ventricular remodeling [1]. Despite progress in acute MI care, novel therapies are critically needed to curb cardiac damage and improve post-MI outcomes in this prevalent condition affecting millions worldwide [2]. Exosomes, nano-sized vesicles secreted by most cell types, have emerged as critical intercellular communicators, ferrying various regulatory molecules like microRNAs (miRNAs) and influencing numerous physiological and pathological processes, including the response to ischemic injury and other aspects of MI pathogenesis [3].

MicroRNAs like miR-21-5p intricately regulate gene levels through silencing target mRNAs and hold important roles in cardiac health and disease [4]. miR-21-5p is enriched in exosomes derived from mesenchymal stem cells (MSCs), a cell type with known cardioprotective paracrine abilities [5]. Delivery of exosomal miR-21-5p via MSC can modulate inflammation, apoptosis, and fibrosis processes that relate with MI [6]. While recent publications have reported that delivering miR-21-5p via exosomes may protect cardiomyocytes and decrease infarct size after ischemia [7], the complete role of miR-21-5p in MI progression and recovery has not been thoroughly investigated.

Yes-associated protein 1 (YAP1), belongs to transcriptional coactivators, is regulated by the Hippo signaling pathway and controls cell proliferation, growth, and survival [8]. Aberrant activation of YAP1 has been implicated in cancer, tissue fibrosis, and other disorders involving cellular damage or death [9]. Bioinformatic analyses predict the presence of miR-21-5p binding sites within the 3’UTR of YAP1 mRNA, suggesting that YAP1 may be the potential target of miR-21-5p [10]. As excessive activation of YAP1 might worsen ischemic injury, the suppression of YAP1 by miR-21-5p may help alleviate damage caused by MI by reducing cardiomyocyte apoptosis and enhancing repair mechanisms.

This study aims to clarify the intricate molecular interaction between exosomal miR-21-5p and YAP1, as well as their potential roles in mitigating MI injury. We will conduct a comprehensive investigation into the delivery of miR-21-5p to cardiomyocytes through exosomes derived from MSCs, as well as its role in suppressing YAP1. This research aims to uncover previously unknown mechanisms of exosomal miRNA regulation that impact the cardiac response to ischemic injury. Exploring the influence of exosomal miR-21-5p on post-MI cardiomyocyte responses, such as inflammation, apoptosis, and fibrotic signaling through YAP1 inhibition, could reveal new therapeutic approaches for improving outcomes after myocardial infarction.

Methods

Cell culture

For our in vitro investigations, rat MSCs (CP-R131) and rat cardiomyocyte H9c2 cells (CL-0089) were procured. MSCs were cultivated in Mesenchymal Stem Cell Growth Medium (CM-R131), while rat cardiomyocyte H9c2 cells were nurtured in Cardiomyocyte Growth Medium (CM-R073). Both cell lines and culture media were acquired from Procell (Wuhan, China) and maintained at 37 °C with a 5% CO₂ atmosphere. MSCs underwent passage when they reached 90% confluence, and cells from the third passage were selected for subsequent experiments.

Characterization of MSCs

To characterize MSCs, we examined the expression of specific cell surface markers. Notably, CD73, CD90, and CD105 exhibited positive expression, while CD14, CD19, and CD34 showed negative expression. The protocol involved washing the cells with phosphate-buffered saline (PBS) and then subjecting them to incubation with primary antibodies against CD14 (PA5-78957), CD19 (MA5-32544), CD34 (PA5-78978), CD73 (PA5-81614), CD90 (MA1-21469), and CD105 (MA1-19231). Subsequently, another incubation was conducted with streptavidin-FITC conjugate (SA10002). These antibodies were sourced from Invitrogen (Carlsbad, CA, USA) and diluted at a ratio of 1:100. Finally, surface marker expression was assessed using a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA).

Isolation and identification of exosomes

The procedures for isolating and characterizing exosomes closely followed previously published methods [11]. In brief, cellular debris was removed by centrifuging the cells at 1500 g for 30 min. The resulting supernatant was then combined with Exosome Isolation Reagent (Invitrogen) at 4 °C for 12 h. Subsequently, the samples underwent centrifugation at 2000 g for 30 min to yield a pellet containing exosomes, which were subsequently suspended in PBS and stored at -80 °C. Exosomal proteins were isolated using the Exosomal Protein Extraction kit (Invitrogen). Western blot was employed to confirm the presence of exosomes by evaluating the protein levels of CD81 (MA5-32333, Invitrogen) and TSG101 (MA5-32463, Invitrogen). Transmission electron microscopy (TEM, Zeiss, Oberkochen, Germany) and nanoparticle tracking analysis (NTA) with a NanoSight LM10 instrument (Malvern Instruments, Malvern, UK) were used to assess the morphology and size distribution of isolated exosomes.

Transmission electron microscopy (TEM)

For TEM characterization, exosome suspensions were prepared by placing a 20 µL aliquot onto a glow-discharged, carbon-coated copper grid (Ted Pella Inc., Redding, CA). After 5 min of adsorption, excess liquid was carefully wicked away using filter paper. The grids were then rinsed twice with distilled water. Subsequently, samples were negatively stained for 2 min using 2% uranyl acetate solution. Following stain removal, the grids were allowed to air-dry completely. Imaging was performed on a Tecnai G2 Spirit BioTWIN transmission electron microscope (FEI Company, Hillsboro, OR) operated at an accelerating voltage of 80 kV. Digital micrographs were acquired using an AMT 2k CCD camera (Advanced Microscopy Techniques, Corp., Woburn, MA).

Nanoparticle tracking analysis (NTA)

The size distribution profile and concentration of isolated exosomes were determined via nanoparticle tracking analysis (NTA) using a NanoSight LM10 instrument (Malvern Instruments, Malvern, UK). Exosome samples were diluted in particle-free phosphate-buffered saline (PBS) to achieve an optimal concentration range for NTA analysis. The diluted suspensions were introduced into the sample chamber and recorded at 25 °C for 60 s, with manual adjustment of the shutter and gain settings. The NTA software tracked and sized individual particles based on their Brownian motion, generating a size distribution profile and corresponding concentration measurement.

Internalization of exosomes

To assess the uptake of exosomes, following the directions on the kit, the isolated exosomes were labeled with PKH26 Red Fluorescent Dye (Mini26, Sigma-Aldrich, St. Louis, MO, USA). In summary, exosomes were incubated at room temperature for five minutes with 2 µM PKH26 dye. The reaction was halted using 1% BSA (Sigma-Aldrich), and excess dye was removed from the exosomes through ultracentrifugation (Optima MAX-X, Beckman, Brea, CA, USA). Labeled exosomes were then incubated with H9c2 cells on glass cover slips (Thermo-Fisher, Waltham, MA, USA) for 24 h. The cells were fixed using 4% paraformaldehyde (Sigma-Aldrich), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich), and the nuclei were counterstained with DAPI (Thermo-Fisher). The results were observed using a fluorescence microscope (Nikon Ti2-E, Tokyo, Japan).

Cell transfection and treatments

To establish gene knockdown models, we obtained a miR-21-5p inhibitor or a YAP1 shRNA plasmid (sh-YAP1) from Genechem, Shanghai, China. As negative controls, randomly scrambled inhibitors (NC) or sh-NC plasmids were utilized for cell transfection. Lipofectamine 3000 (Thermo-Fisher) facilitated the transfection process.

For cell treatments, MSCs were categorized into three groups: Control group, miR-21-5p inhibitor group, and inhibitor NC group. The exosomes derived from these groups were collected as Exos group, Exos-inhibitor group, and Exos-NC group, respectively, and were employed for subsequent experiments. To establish in vitro models of myocardial infarction (MI), H9c2 cells underwent a series of treatments, including exposure to exosomes from various MSC groups or transfection with sh-YAP1 or sh-NC. Following a 48-h incubation period, the cells were exposed to hypoxia by culturing them in a modular incubator chamber with an atmosphere of 1% O2, 5% CO2, and 94% N2 for 24 h. Subsequently, H9c2 cells were harvested for further biological analyses.

Quantitative real-time PCR (RT-qPCR)

Total RNA extraction from cells was carried out using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. A NanoDrop 2000 (Thermo-Fisher) was employed to measure RNA concentration. The PrimeScript RT Reagent Kit (Takara, Tokyo, Japan) was used to reverse transcribe the isolated total RNA into cDNA. This RT-qPCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System with SYBR Premix Ex Taq II (Takara). The cycling conditions included an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. GAPDH served as an internal control. Relative mRNA expression levels were calculated using the 2-ΔΔCt method. The primer sequences are provided in Table S1.

Western blot

RIPA buffer (Thermo-Fisher), which contains protease and phosphatase inhibitors, was used for cell lysis. A BCA assay kit was used to determine the protein concentration (Solarbio, Beijing, China). Subsequently, 30 µg of protein underwent separation via 10% SDS-PAGE and was transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 1 h, membranes underwent incubation with primary antibodies against CD18 (MA1-10122), TSG101 (MA5-32463), Bax (MA5-14003), cleaved caspase-3 (PA5-114687), cleaved caspase-9 (PA5-105271), Bcl-2 (PA5-27094), YAP1 (PA1-46189), GAPDH (MA1-16757) at 4 °C overnight. Following washing, the membranes were probed with HRP-conjugated secondary antibodies (31460, 1:1000) for 1 h at room temperature. These antibodies were sourced from Invitrogen (Carlsbad, CA, USA) and diluted at a ratio of 1:1000. Protein bands were visualized using an ECL substrate kit (Millipore) and quantified using ImageJ software.

MTT assay

To evaluate cell viability, an MTT assay was conducted. In brief, 5 × 10³ cells per well were seeded in 96-well plates. Subsequently, 20 µl of MTT solution (Solarbio) was introduced into each well and incubated at 37 °C for 4 h. The formazan crystals formed were then dissolved in 150 µl of DMSO. The microplate reader (BioTek, Shoreline, WA, USA) was employed to detect absorbance at 570 nm.

ELISA measurement

Commercial ELISA kits (Solarbio) were employed for monitoring the levels of TNF-α, IL-1β, and IL-6 in the supernatant of culture medium or animal tissues, following the manufacturer’s protocols. Absorbance readings were taken at 450 nm by a microplate reader (BioTek), and cytokine concentrations were determined through standard curve calculations.

Flow cytometric analysis of cell apoptosis

Cell apoptosis was ascertained utilizing the Annexin V-FITC/PI apoptosis detection kit (BD Biosciences). In brief, the cells underwent harvesting, followed by a cold PBS wash and suspension in 1X binding buffer. Subsequently, cells were subjected to staining with 5 µl of Annexin V-FITC and 5 µl of propidium iodide (PI) for 15 min in darkness. Quantification of apoptotic cells was performed using a FACS Canto II flow cytometer (BD Biosciences). FlowJo software was utilized to analyze early apoptotic cells (Annexin V-positive, PI-negative) and late apoptotic cells (Annexin V-positive, PI-positive).

Microarray profiling of miRNA expression

Total RNA containing small RNAs was extracted from cellular specimens employing the miRNeasy Mini Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s recommended protocol. An assessment of RNA quality was carried out using a Bioanalyzer (Agilent), with samples boasting a RIN > 7 being deemed suitable for further analysis. miRNA underwent labeling via the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA, USA). The labeled miRNA was subsequently subjected to hybridization on Affymetrix GeneChip miRNA 4.0 arrays, maintained at 48 °C for a duration of 16 h. After hybridization, the arrays underwent washing and staining procedures using a Fluidics Station 450, culminating in scanning on a GeneChip Scanner 3000 via the Command Console software (Affymetrix). The raw data underwent normalization through the robust multi-array average method, facilitated by the Transcriptome Analysis Console software (Affymetrix). Differential expression of miRNAs was filtered based on criteria encompassing a fold change of ≥ 1.5 and a p-value of < 0.05.

Dual luciferase reporter assay

The pmirGLO dual luciferase vector (Promega, Madison, WI, USA) was employed to synthesize and insert the YAP1 3’UTR, containing the presumed binding site for miR-21-5p. Mutations within the seed region were introduced using the QuikChange Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA). Utilizing Lipofectamine 3000 (Invitrogen), H9c2 cells underwent co-transfection with either the miR-21-5p mimic or inhibitor along with the luciferase reporter plasmids. After a 24-h incubation period, with the use of the Dual-Luciferase Reporter Assay System (Promega), the activities of firefly and renilla luciferases were evaluated. The internal control was provided by Renilla luciferase.

RNA immunoprecipitation (RIP)

Cell lysates, as mentioned earlier, were subjected to incubation with protein A/G magnetic beads (Thermo-Fisher) conjugated with either anti-Ago2 antibody or control IgG, all performed at 4 °C overnight with rotation. Subsequently, beads underwent washing, and quantification of miR-21-5p levels that had been immunoprecipitated was carried out using RT-qPCR, as previously described. MiR-21-5p levels in Ago2 immunoprecipitates were compared to those in IgG immunoprecipitates.

Animal models

All animal experiments were approved by the Laboratory Animals Ethics Committee of Jinzhou Medical University (No. SYXK2019-0007) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council). A total of 40 male Sprague-Dawley rats (200–250 g, 8–10 weeks old) were obtained and housed in a temperature-controlled environment with a 12-hour light/dark cycle and access to food and water ad libitum.

Rats were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg). After endotracheal intubation, rats were mechanically ventilated. Left thoracotomy was performed in the fourth intercostal space to expose the heart. Acute myocardial infarction (AMI) was induced by permanent ligation of the left anterior descending (LAD) coronary artery using a 7 − 0 polypropylene suture. Successful LAD occlusion was confirmed by myocardial blanching and electrocardiographic ST-segment changes. Sham-operated animals underwent the same surgical procedure without LAD ligation.

One-week post-surgery, rats were randomized into four groups (n = 10 per group): (1) Sham, (2) AMI, (3) Control-Exo, (4) miR-21-5p-Exo group. The Exosome group received 100 µg of exosomes suspended in 100 µL sterile phosphate-buffered saline (PBS) via tail vein injection. Sham and MI groups received equal volumes of PBS. After 4 weeks, rats were euthanized by exsanguination under anesthesia. Hearts were harvested, washed in cold PBS, and either snap-frozen for molecular analysis or fixed in 10% neutral-buffered formalin for histological studies.

Masson’s staining

Formalin-fixed paraffin heart Sect. (5 μm) underwent deparaffinization in xylene and rehydration through graded ethanol. Sections were mordanted in pre-heated Bouin’s solution at 55 °C for 15 min, followed by washing under running tap water. Weigert’s iron hematoxylin staining was performed for 5 min to label nuclei. After rinsing, sections were stained with Biebrich scarlet-acid fuchsin solution for 5 min to detect cytoplasm and muscle fibers. Sections were differentiated in phosphomolybdic-phosphotungstic acid solution, and subsequently transferred to aniline blue solution for 5 min to visualize collagen fibers. Finally, sections were rinsed in 1% acetic acid, dehydrated through ethanol, cleared in xylene, and mounted. Digital slide scanning and automated image analysis were performed to determine infarct area.

Immunohistochemistry

Paraffin sections were deparaffinized and rehydrated, followed by heat-induced antigen retrieval in citrate buffer (pH 6.0) for 20 min at 98 °C. Endogenous peroxidases were quenched by incubating sections in 3% hydrogen peroxide for 10 min. After blocking with protein block solution, sections were incubated overnight at 4 °C with rabbit anti-Cleaved Caspase-3 antibody (#9661, Cell Signaling Technology, 1:400 dilution). The next day, sections were washed and incubated with ImmPRESS HRP anti-rabbit IgG polymer detection reagent (Vector Laboratories) for 30 min at room temperature. Signal development was achieved using ImmPACT DAB peroxidase substrate (Vector Laboratories) for 2 min, followed by counterstaining with hematoxylin. Whole slide imaging and quantitative analysis were performed to determine the percentage of cleaved caspase-3 positive cells.

TUNEL assay

The Click-iT Plus TUNEL assay (Invitrogen) was employed to detect DNA fragmentation in paraffin-embedded heart sections, according to the supplier’s recommendations. In brief, after deparaffinization and rehydration, sections were incubated with Proteinase K (20 µg/ml) for 15 min at room temperature to permeabilize tissues. Endogenous peroxidases were inactivated by treating sections with 3% hydrogen peroxide for 10 min. Sections were then incubated with TdT reaction mixture for 60 min at 37 °C to label DNA strand breaks. The reaction was terminated, and sections were incubated with Click-iT Plus TUNEL reaction cocktail for 30 min at 37 °C to detect incorporated EdUTP. Signals were developed using the Click-iT Plus TUNEL HRP detection kit, followed by DAB substrate addition. Finally, sections were counterstained with methyl green. Fluorescent imaging and automated analysis quantified the percentage of TUNEL-positive nuclei relative to total nuclei.

Statistical analysis

The data presented represents the mean ± standard deviation (SD) of no less than three independent experiments. Statistical comparisons between two groups were executed utilizing an unpaired two-tailed Student’s t-test. For comparisons involving multiple groups, a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was employed. A p-value below 0.05 was regarded as statistically significant. All statistical analyses were conducted using GraphPad Prism 8 software.

Results

Isolation and characterization of MSCs and MSC-derived exosomes

The visual assessment in Fig. 1A revealed the fibroblast-like morphology of MSCs as observed under light microscopy. Flow cytometry analysis, as depicted in Fig. 1B, confirmed the positive expression of surface markers CD105, CD73, and CD90, while markers CD34, CD14, and CD19 were distinctly absent. The examination of isolated exosomes (Fig. 1C) exhibited their characteristic cup-shaped morphology through TEM. Furthermore, NTA demonstrated that these exosomes fell within a diameter range of 30–140 nm (Fig. 1D). Western blot analysis, illustrated in Fig. 1E, clearly indicated an enriched expression of exosomal markers CD81 and TSG101 when compared to MSCs themselves. To explore the internalization of exosomes by H9c2 cardiomyocytes, exosomes were labeled with PKH26 dye before co-incubation. The results (Fig. 1F) vividly show a increase in PKH26-positive exosomes within the cytoplasm of H9c2 cells, providing compelling evidence of exosome internalization via endocytosis. In summary, these findings collectively establish the successful isolation of MSCs and exosomes, their verified identity, and the effective uptake of exosomes by recipient cardiomyocytes.

Fig. 1.

Isolation and characterization of MSCs and MSC-derived exosomes. (A) MSCs morphology observed under light microscopy. (B) MSC surface markers analyzed by flow cytometry. (C) Exosome morphology examined by TEM. (D) Exosome size distribution measured by NTA. (E) Exosomal markers CD81 and TSG101 measured in MSCs and exosomes by Western blot. (F) Exosome uptake by H9c2 cells visualized after PKH26 labeling by confocal microscopy. All experiments were performed in three independent biological replicates

Exosomes inhibit inflammation and apoptosis in hypoxic H9c2 cells

The impact of hypoxia on H9c2 cell viability was found to be significant, resulting in a notable decline. However, treatment with exosomes ameliorated this effect, as depicted in Fig. 2A. Inflammatory factors, namely TNF-α, IL-1β, and IL-6, exhibited a considerable surge following exposure to hypoxia. Remarkably, the introduction of exosomes led to a substantial decrease in the expression of these cytokines (Fig. 2B). Furthermore, hypoxia exerted a substantial pro-apoptotic influence on H9c2 cells, resulting in an increased rate of apoptosis. Conversely, the presence of exosomes effectively mitigated apoptosis (Fig. 2C) At the molecular level, hypoxia prompted an upregulation of pro-apoptotic proteins such as Bax, cleaved caspase-3, and cleaved caspase-9, while concurrently downregulating the anti-apoptotic protein Bcl-2. Interestingly, the introduction of exosomes reversed these trends, as demonstrated in Fig. 2D. Collectively, these findings underscore the capacity of MSC-derived exosomes to counteract hypoxia-induced inflammation and apoptosis in H9c2 cardiomyocytes.

Fig. 2.

Exosomes inhibit inflammation and apoptosis in hypoxic H9c2 cells. (A) Cell viability measured by MTT assay. (B) Inflammatory cytokines measured by ELISA. (C) Apoptosis analyzed by flow cytometry. (D) Apoptosis-related proteins measured by Western blot. All experiments were performed in three independent biological replicates. *P < 0.05,**P < 0.01,***P < 0.001

Exosomes significantly increase mir-21-5p expression in H9c2 cells

Upon microarray analysis, it became evident that exosomes exerted a considerable influence on miRNA expression within H9c2 cells, resulting in the notable upregulation of eight key miRNAs, specifically miR-706, miR-155, miR-486-5p, miR-708-3p, miR-93-5p, miR-21-5p, miR-133a-3p, and miR-125b (Fig. 3A). Notably, these miRNAs were found to be more abundant in exosomes when compared to the MSC conditioned medium, with miR-21-5p exhibiting the highest enrichment (Fig. 3B). The effective manipulation of miR-21-5p expression levels within MSCs, achieving both overexpression and knockdown, was duly confirmed (Fig. 3C). Furthermore, it was observed that exosomes derived from miR-21-5p knockdown MSCs (referred to as Exos-inhibitor) contained notably lower levels of miR-21-5p in contrast to control exosomes (Fig. 3D). In summation, these findings collectively establish the significant role of exosomes in inducing a substantial upregulation of miR-21-5p levels within recipient H9c2 cardiomyocytes.

Fig. 3.

Exosomes increase miR-21-5p levels in H9c2 cells. (A) miRNA microarray of H9c2 cells with or without exosome treatment. (B) miRNA levels measured by qRT-PCR. (C) miR-21-5p overexpression and knockdown efficiency in MSCs. (D) miR-21-5p levels in control vs. inhibitor exosomes. All experiments were performed in three independent biological replicates. *P < 0.05,**P < 0.01,***P < 0.001

Inhibiting mir-21-5p diminishes the protective effects of exosomes in hypoxic H9c2 cells

As presented in Fig. 4A, the knockdown of miR-21-5p within exosomes (Exos-inhibitor) contributed to a reduction in miR-21-5p levels within H9c2 cells. This decrease in miR-21-5p levels corresponded with a diminished cell viability when compared with the control exosome group, as evidenced in Fig. 4B. Concomitantly, the treatment with Exos-inhibitor resulted in an upsurge in the expression of inflammatory factors, including TNF-α, IL-1β, and IL-6 (Fig. 4C). These findings underline the influence of Exos-inhibitor in exacerbating inflammatory responses within the H9c2 cells. Furthermore, flow cytometry analyses illustrated that the suppression of miR-21-5p contributed to an increased rate of apoptosis within the H9c2 cells (Fig. 4D). This increase in apoptosis was associated with elevated levels of pro-apoptotic proteins, such as Bax, cleaved caspase-3, and cleaved caspase-9, alongside a reduction in the levels of anti-apoptotic Bcl-2 (Fig. 4E). The above findings underscore the pivotal role of miR-21-5p in mediating the anti-inflammatory and anti-apoptotic effects of exosomes, as the inhibition of miR-21-5p within exosomes resulted in the attenuation of their protective impact in hypoxic H9c2 cells.

Fig. 4.

Inhibiting exosomal miR-21-5p reduces protection in hypoxic H9c2 cells. (A) miR-21-5p levels measured by qRT-PCR. (B) Cell viability by MTT. (C) Inflammatory cytokines by ELISA. (D) Apoptosis by flow cytometry. (E) Apoptosis-related proteins by Western blot. All experiments were performed in three independent biological replicates. *P < 0.05,**P < 0.01,***P < 0.001

Mir-21-5p directly targets and inhibits YAP1

A computational analysis revealed the presence of a putative binding site for miR-21-5p within the 3’UTR of YAP1, as depicted in Fig. 5A. Intriguingly, the introduction of miR-21-5p inhibitor led to a discernible increase in luciferase activity when the YAP1-3’UTR (YAP1-Wt) was present, while the mutant variant (YAP1-Mut) remained unaffected (Fig. 5B). Furthermore, the results of RIP unveiled a notable enrichment of both miR-21-5p and YAP1 within Ago2, in stark contrast to the IgG control (Fig. 5C), thus affirming their interaction. In the context of hypoxia, it became evident that both YAP1 mRNA and protein levels experienced an elevation, a phenomenon effectively mitigated by the presence of exosomes. Conversely, the application of Exos-inhibitor resulted in a conspicuous enhancement in YAP1 level relative to the control exosome group, as delineated in Fig. 5D&E. In summary, these collective findings robustly substantiate miR-21-5p’s direct binding affinity with the YAP1 3’UTR, leading to a consequential inhibition of its expression. Moreover, the mechanism of exosomal transfer of miR-21-5p emerges as a potent means to suppress YAP1 within the context of hypoxic H9c2 cells.

Fig. 5.

miR-21-5p directly targets and inhibits YAP1. (A) Bioinformatic prediction of miR-21-5p binding site. (B) Dual luciferase reporter assay. (C) RNA immunoprecipitation. (D) YAP1 mRNA levels by qRT-PCR. (E) YAP1 protein levels by Western blot. All experiments were performed in three independent biological replicates

Exosomes act through the miR-21-5p/YAP1 axis in hypoxic H9c2 cells

The knockdown of YAP1 had a notable impact, leading to decreased levels of YAP1 mRNA without affecting miR-21-5p levels (Fig. 6A). This decrease in YAP1 level was associated with increased cell viability (Fig. 6B), reduced levels of inflammatory cytokines (Fig. 6C), diminished apoptosis (Fig. 6D), and alterations in apoptosis-related proteins (Fig. 6E) within Exos-inhibitor treated cells. Consequently, the reduction of YAP1 expression in cells with inhibited exosomal miR-21-5p was found to effectively restore the protective effects on hypoxic H9c2 cells. These findings underscore the pivotal role of exosomes in mediating their effects through the miR-21-5p/YAP1 axis, thereby impacting the processes of inflammation and apoptosis.

Fig. 6.

Exosomes act via miR-21-5p/YAP1 in hypoxic H9c2 cells. (A) YAP1 and miR-21-5p levels by qRT-PCR after YAP1 knockdown. (B) Cell viability by MTT assay. (C) Inflammatory cytokines by ELISA. (D) Apoptosis by flow cytometry. (E) Apoptosis proteins by Western blot after YAP1 knockdown. All experiments were performed in three independent biological replicates. *P < 0.05,**P < 0.01,***P < 0.001

Exosomal mir-21-5p inhibits apoptosis and preserves cardiac function in a rat model of myocardial infarction

Masson’s trichrome staining revealed that miR-21-5p-Exo treatment significantly reduced infarct size and collagen deposition compared to AMI and Ctrl-Exo groups (Fig. 7A). TUNEL and cleaved caspase-3 immunohistochemistry demonstrated that exosomal miR-21-5p potently inhibited cardiomyocyte apoptosis in the infarct border zones, with the miR-21-5p-Exo group exhibiting the lowest number of TUNEL + and cleaved caspase-3 + cells (Fig. 7B). Furthermore, ELISA analysis showed that miR-21-5p-Exo treatment markedly reduced myocardial levels of the pro-inflammatory cytokines IL-6 and TNF-α compared to control groups (Fig. 7C). Western blot analysis confirmed that miR-21-5p-Exo administration significantly decreased YAP1 protein expression in the infarcted hearts (Fig. 7D). Collectively, these results indicate that exosomal transfer of miR-21-5p confers cardioprotection against MI injury by inhibiting apoptosis, inflammation, and YAP1 signaling in vivo.

Fig. 7.

miR-21-5p-Exosomes attenuate apoptosis and preserve cardiac function in a rat myocardial infarction model. (A) Masson’s Trichrome staining of heart sections showing infarct area (blue staining). (B) TUNEL and cleaved caspase-3 staining demonstrating cell apoptosis. (C) ELISA analysis revealing expression of IL-6 and TNF-α. (D) Western blotting analysis showing YAP1 expression. N = 10 rats in each group. *P < 0.05,**P < 0.01,***P < 0.001

Discussion

Extracellular vesicles derived from mesenchymal stem cells (MSCs) have emerged as vital paracrine messengers, offering a promising avenue for significantly enhancing outcomes in the context of ischemic injury. Recent research has underscored the therapeutic potential of umbilical cord MSC-derived exosomes (UMSC-Exo) [12]. They have been shown to ameliorate muscle function by delivering circHIPK3, effectively curtailing ischemia-induced pyroptosis, and facilitating the repair of ischemic muscle injuries [12]. This study added to the growing body of evidence supporting exosome-mediated delivery of miRNAs as a noteworthy approach in cardiovascular therapy. Multiple investigations have attested to exosomes’ potential role as a therapeutic intervention for acute myocardial infarction (AMI) [13]. For instance, Mao et al. highlighted the exosomal lncRNA KLF3-AS1, secreted by human MSCs, which acts as a sponge for miR-138-5p, thereby regulating Sirts [14]. This mechanism results in reduced cellular apoptosis and pyroptosis, thereby mitigating MI progression. Furthermore, a study brought to light the positive impact of FNDC5 pre-conditioning in bone marrow-derived MSCs, which effectively reduced pro-inflammatory secretion and dampened the NF-κB signaling pathway in the context of MI [15]. Our own research findings align with these reported benefits, underscoring that MSC-derived exosomes robustly suppress inflammation and apoptosis in hypoxic cardiomyocytes.

Several studies have underscored the protective roles of miR-21-5p in the setting of MI. Notably, miR-21-5p levels tend to be upregulated in the post-ischemic heart [16]. The augmentation of miR-21-5p expression, either through mimics or exosome-based delivery, has consistently demonstrated a reduction in infarct size and the preservation of cardiac function in AMI animal models [17]. A key underlying mechanism for these benefits is the ability of miR-21-5p to inhibit programmed cell death 4 (PDCD4), thereby suppressing apoptosis in vulnerable cardiomyocytes following MI [18]. Additional research suggests that miR-21-5p may also attenuate inflammation after ischemic injury by downregulating NF-κB signaling [19]. It holds promise in limiting cardiac fibrosis by targeting factors such as SPRY-1, although further investigations are warranted to elucidate its complete anti-fibrotic capabilities [20]. Moreover, circulating miR-21-5p exhibits potential as a plasma biomarker for early MI diagnosis and prognosis [21]. In summation, these studies underscore miR-21-5p as a pivotal mediator of the cardiac response to ischemic injury. Harnessing the cardioprotective effects of miR-21-5p through miRNA mimics or exosomal delivery presents a promising therapeutic strategy for mitigating myocardial damage post-MI. Consistently, our current study affirms that miR-21-5p enhances cell viability, ameliorates the inflammatory status, and reduces cell apoptosis in the context of MI.

Recent investigations have spotlighted the role of YAP1 activation in cardiac fibroblasts post-MI. YAP1 promotes myofibroblast differentiation, inflammatory cytokine production (e.g., IL-33), and excessive extracellular matrix deposition, which collectively contribute to detrimental ventricular fibrosis and impaired cardiac function [22]. Furthermore, YAP1 fosters pro-inflammatory polarization of infiltrating macrophages at the infarct site, intensifying inflammation and hampering the healing process [23]. Li et al. reported that YAP1 could upregulate α-SMA, CTGF and Collagen I, which were key factors for myofibroblast formation and progression of MI [24]. Inhibiting YAP1 signaling, on the other hand, mitigates fibrotic remodeling, enhances anti-inflammatory macrophage responses, and ultimately leads to improved outcomes post-MI [23]. Our study not only highlights the direct binding interactions between miR-21-5p and YAP1 but also reveals that the protective roles of miR-21-5p are mediated through its targeting of YAP1 in the context of MI.

In a comprehensive perspective, our findings from this study highlight the importance of the miR-21-5p/YAP1 regulatory axis in the pathogenesis of MI and identify it as a potential therapeutic target. Dysregulated YAP1 signaling has been implicated in driving pathological processes such as cardiomyocyte apoptosis, fibroblast activation, and adverse ventricular remodeling following ischemic injury. Therefore, targeted inhibition of YAP1 by miR-21-5p or other approaches could represent a promising strategy to preserve cardiac structure and function in MI. Furthermore, the ability of exosomes to efficiently deliver functional miRNAs to target cells holds significant potential for RNA-based therapies in cardiovascular disease. Exosome-mediated delivery of miR-21-5p may enhance targeted delivery to the ischemic myocardium while minimizing systemic toxicity, thereby maximizing therapeutic efficacy. Nevertheless, there are some limitations to the current study. The rationale for initially selecting YAP1 as a downstream target of miR-21-5p could be more comprehensively justified, and the downstream signaling pathways of the Hippo-YAP1 axis warrant further exploration. Additionally, the long-term effects, stability, and potential off-target interactions of exosomal miR-21-5p delivery remain to be elucidated. Future studies should evaluate the pharmacokinetics, biodistribution, clearance kinetics, and feasibility of repeated dosing regimens for exogenously administered exosomes. Genome-wide transcriptomic and proteomic analyses would also provide valuable insights into the specificity of miR-21-5p targeting and its broader impacts on cellular pathways beyond the YAP1 axis.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Zhou Ji: Conceptualization, Project administration, Investigation, Resources, Validation, Formal analysis, Methodology, Writing - Original Draft and Writing - Review & Editing. Chan Wang: Conceptualization, Data Curation, Visualization and Writing - Review & Editing. All authors read and approved the final manuscript.

Funding

This study was funded by the Natural Science Foundation of Liaoning Provincial Department of Education (No. LJKMZ20221252). Joint Fund of Liaoning Provincial Science and Technology Department and Jinzhou Medical University Project (No. 2023-BSBA-152).

Data availability

All data generated or analysed during this study are included in this article.

Declarations

Ethical approval

All animal experiments were approved by the Laboratory Animals Ethics Committee of Jinzhou Medical University (No. SYXK2019-0007). All experiments are reported in accordance with ARRIVE guidelines.

Competing interests

The authors declare no competing interests.