Role of Myocardium-Derived Exosomal miRNAs in Doxorubicin-Induced Cardiomyopathy

Role of Myocardium-Derived Exosomal miRNAs in Doxorubicin-Induced Cardiomyopathy | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 7 August 2025 V1 Latest version Share on Role of Myocardium-Derived Exosomal miRNAs in Doxorubicin-Induced Cardiomyopathy Authors : Cui Li 0009-0007-8701-573X , Yudie Song , Binbin Cao , Jiahui Li , Fan Xu , Weiping Du , Zhaoxia Zhang , Xiaomin Chen , Qinglin Yu , and Jia Su 0000-0003-2163-2739 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175458092.25863622/v1 Published Gene Version of record Peer review timeline 150 views 98 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Doxorubicin-induced cardiomyopathy (DCM) remains a major clinical challenge, highlighting the need for early diagnostic biomarkers. Exosomal miRNAs have emerged as promising candidates because of their stability and regulatory roles in cardiovascular diseases. Methods: Exosomes were isolated from myocardial tissue samples of DOX-treated and control rats using differential ultracentrifugation and were subsequently characterized by transmission electron microscopy (TEM), Western blotting, and nanoparticle tracking analysis (NTA). Differentially expressed miRNAs were screened by microarray analysis. The miRanda algorithm was used to predict target genes, followed by functional enrichment analysis. qRT‒PCR validated selected miRNAs and their target genes. Results: Doxorubicin administration induced significant cardiac dysfunction, characterized by decreased left ventricular ejection fraction (LVEF) and increased serum levels of cardiac biomarkers (cTnI, BNP, and CK-MB). Microarray profiling revealed 26 differentially expressed miRNAs, and KEGG enrichment analysis indicated enrichment in the MAPK signaling pathway. qRT‒PCR demonstrated upregulation of exosomal miR-182 and downregulation of Taok2 mRNA, a key MAPK gene. ROC analysis indicated the potential of exosomal miR-182 for DCM diagnosis. Conclusion: Exosomal miR-182 may serve as a diagnostic marker for DCM. Bioinformatic analyses predict its potential regulation of the MAPK pathway through Taok2 targeting, although this mechanistic relationship requires experimental validation. These findings provide new perspectives for the early monitoring of DCM. \received DD MMMM YYYY \acceptedDD MMMM YYYY Role of Myocardium-Derived Exosomal miRNAs in Doxorubicin-Induced Cardiomyopathy Cui Li a,b,1 , Yudie Song a,b,1 ,Binbin Cao a,b,1 ,Jiahui Li a,b ,Fan Xu a,b ,Weiping Du a,b , Zhaoxia Zhang a,b ,Xiaomin Chen a,b, ,Qinglin Yu c,* , Jia Su a,b,* a Department of Cardiology, The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China b Key Laboratory of Precision Medicine for Atherosclerotic Diseases of Zhejiang Province, China c Department of Traditional Chinese Internal Medicine, The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China 1 Equal contributors:Cui Li,Yudie Song,Binbin Cao are the Equal contributors to this article and they are first co-authors. * Corresponding author:Jia Su( [email protected] )and QinglinYu( [email protected] ) are addressd as corresponding author. Keywords:Cardio-oncology,Doxorubicin-induced cardiomyopathy, Myocardium-Derived Exosomal miRNAs, miR-182,Taok2, Abstract Background: Doxorubicin-induced cardiomyopathy (DCM) remains a major clinical challenge, highlighting the need for early diagnostic biomarkers. Exosomal miRNAs have emerged as promising candidates because of their stability and regulatory roles in cardiovascular diseases. Methods: Exosomes were isolated from myocardial tissue samples of DOX-treated and control rats using differential ultracentrifugation and were subsequently characterized by transmission electron microscopy (TEM), Western blotting, and nanoparticle tracking analysis (NTA). Differentially expressed miRNAs were screened by microarray analysis. The miRanda algorithm was used to predict target genes, followed by functional enrichment analysis. qRT‒PCR validated selected miRNAs and their target genes. Results: Doxorubicin administration induced significant cardiac dysfunction, characterized by decreased left ventricular ejection fraction (LVEF) and increased serum levels of cardiac biomarkers (cTnI, BNP, and CK-MB). Microarray profiling revealed 26 differentially expressed miRNAs, and KEGG enrichment analysis indicated enrichment in the MAPK signaling pathway. qRT‒PCR demonstrated upregulation of exosomal miR-182 and downregulation of Taok2 mRNA, a key MAPK gene. ROC analysis indicated the potential of exosomal miR-182 for DCM diagnosis. Conclusion: Exosomal miR-182 may serve as a diagnostic marker for DCM. Bioinformatic analyses predict its potential regulation of the MAPK pathway through Taok2 targeting, although this mechanistic relationship requires experimental validation. These findings provide new perspectives for the early monitoring of DCM. 1 Introduction As a prominent member of the anthracycline class of chemotherapeutic agents, Doxorubicin（DOX） exhibits potent antitumor activity and remains a cornerstone in the treatment of multiple malignancies, particularly breast carcinoma and leukemic disorders [1] . However, its clinical utility is significantly limited by dose-dependent cardiotoxicity, characterized by myocardial accumulation leading to ventricular dilation, progressive cardiac dysfunction, and ultimately heart failure [2] . Despite decades of research, dexrazoxane remains the only FDA-approved cardioprotectant, and its application is limited by myelosuppression risk [3] . These challenges underscore the urgent need for novel therapeutic strategies. MicroRNAs (miRNAs), which are 19–23 nt noncoding RNAs that silence target genes through 3’-UTR binding, are pivotal regulators of cardiovascular diseases [4] . Their dysregulation contributes to diverse pathologies, including heart failure and drug-induced cardiotoxicity [5] . Notably, DOX treatment rapidly elevates plasma miR-1/miR-499 levels within 24 hours [4] , whereas miR-212/132 overexpression has cardioprotective effects by reducing apoptosis and improving ventricular function [6] . These findings collectively validate the potential of miRNAs in cardiovascular medicine for diagnosis and treatment, with several candidates already transitioning into clinical applications. Exosomes, a key subtype of extracellular vesicles secreted by diverse cell types, feature a phospholipid bilayer structure that enables the transport of proteins, nucleic acids, and other biomolecules for intercellular communication [7] . Therefore, exosomes can indicate physiological or pathological states and hold significant value in disease diagnosis. For example, studies have shown that, compared with those in healthy controls, serum exosomal miR-21-5p and miR-375-5p are elevated in patients with type 1 diabetes mellitus [8] , whereas increased exosomal CD93 and inflammatory cytokines (IFN-γ, TNF-α, and IL-4) are correlated with HIV-associated cryptococcal meningitis [9] . In osteosarcoma, the levels of plasma exosomal CD63, vimentin and epithelial cell adhesion molecules are markedly increased, with high diagnostic specificity [10] . Notably, exosomal TSPAN1 has superior diagnostic performance over conventional CEA biomarkers in hepatocellular carcinoma [11] . Exosomal miRNAs have emerged as highly promising research tools because of their remarkable modifiability, with unique advantages, including efficient penetration through cellular membranes and the blood‒brain barrier to enable both local and distant target cell regulation by signaling pathways [12] . Furthermore, their characteristic lipid bilayer structure effectively protects miRNAs from extracellular enzymatic degradation, ensuring structural integrity and stability during transport [13] . Although the role of exosomal miRNAs in cardiovascular diseases has been explored, studies focusing specifically on myocardial-derived exosomes in DCM remain limited. These exosomes reflect cardiomyocyte-specific alterations more accurately than it does those derived from serum or plasma. In this study, we used microarray technology to analyze the myocardial exosomal miRNA expression profiles in a DCM rat model and identified several potential diagnostic markers. These findings offer new insights for the early diagnosis and targeted treatment of DCM. 2 Methods The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies [14] . 2.1 Animals and Treatment All procedures involving animal experimentation were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals (NIH, revised 2011). Male Sprague–Dawley (SD) rats weighing 200–250 g were obtained from the Chinese Academy of Medical Sciences and utilized in this study. Prior to the experimental manipulation, the rats were acclimatized for 3 days in a constant-temperature (20–25 °C) environment with a 12-h light/dark cycle with illumination from 7:00 AM to 7:00 PM, during which they had free access to water and food. The rats were randomly divided into a model group and a control group (n=12 per group). The model group was administered 2.5 mg/kg DOX via intraperitoneal injection every 48 hours, whereas the control group received an equivalent volume of normal saline. This dosing regimen was maintained for a total duration of 16 days. Serum concentrations of cardiac injury biomarkers, including cardiac troponin I (cTnI), B-type natriuretic peptide (BNP), and creatine kinase-MB (CK-MB), were quantified within 24 hours after collection by enzyme-linked immunosorbent assay (ELISA). 2.2 Detection of Cardiac Function and Myocardial Injury Markers Sixteen days after administration, 3 rats from each group were randomly selected for cardiac ultrasound examination and serum marker analysis. Prior to the procedures, the rats were anesthetized by the intraperitoneal administration of 1.5% pentobarbital sodium (3 mL/kg) and fixed in the supine position. Transthoracic echocardiography was performed to evaluate the following cardiac function parameters: LVEF, fractional shortening (LVFS), end-diastolic internal diameter (LVIDd), and end-systolic internal diameter (LVIDs). Immediately after the cardiac ultrasound examination, cardiac blood samples were collected. Following heparin anticoagulation, the samples were centrifuged (10,000 ×g, 5 min, 4 °C) to isolate the serum, which was subsequently stored at −80 °C until analysis. 2.3 Myocardial sample preparation and exosome isolation Cardiac tissues were collected separately from experimental groups 1−5 and control groups 1−5 (approximately 1.2 g per sample). Tissue processing involved enzymatic digestion using 5 mL of collagenase IV (2 mg/mL) at 37 °C for 60 minutes. Following digestion, the samples were subjected to differential centrifugation, first at 300×g for 10 minutes to remove intact cells and then at 2,000×g for 20 minutes to eliminate cellular debris. After the supernatant was collected and the pellet was discarded, exosome isolation was achieved through ultracentrifugation at 20,000×g for 30 minutes. 2.4 Exosome characterization 2.4.1 Transmission electron microscopy Ten microliter aliquots of exosomes were applied to Formvar-coated copper grids (200 mesh). The grids were first immobilized by treatment with 50 μl of 1% (w/v) glutaraldehyde solution for 5 minutes, followed by three washes with purified water. Negative staining was performed using 4% (w/v) uranyl acetate aqueous solution for 3 minutes. The grids were air-dried in a desiccator for several minutes. Exosome morphology was examined using a JEOL JEM-2100 transmission electron microscope (Tokyo, Japan) operating at 80 kV, with images captured at 44,000× magnification (100 nm). 2.4.2 Western blot analysis Exosome proteins were solubilized in RIPA lysis buffer and quantified using the Bradford method (Bio-Rad). Approximately 15 μg of protein was separated by 10% SDS‒PAGE and subsequently transferred to 0.45 μm PVDF membranes (Millipore) using standard wet transfer protocols. The membranes were incubated in TBST containing 5% bovine serum albumin for 90 minutes to prevent nonspecific binding, after which they were incubated with primary antibodies (Alix, TSG101, CD63, CD9, and calnexin; Santa Cruz Biotechnology, CA) overnight at 4 °C. Afterward, the membrane was treated with a secondary antibody solution for 1 hour at 4 °C. Protein detection was performed using an enhanced chemiluminescence system (NCMP10300) and visualized by a ChemiDoc MP system (Bio-Rad). 2.4.3 Nanoparticle tracking analysis Exosome characterization was performed using nanoparticle tracking analysis (NanoSight LM10, Malvern Panalytical, UK) according to standardized protocols. The isolated exosomes were first diluted tenfold in filtered PBS prior to being added to the measurement chamber. NTA was performed with a NanoSight LM10 system equipped with a blue laser (405 nm). The laser illuminated the nanoparticles, and their Brownian motion was recorded for 60 seconds in three separate videos. The videos were analyzed using NTA software to calculate the exosome concentration, size distribution, and other metrics. 2.5 Total RNA extraction from the exosomes First, the sample was lysed with TRIzol reagent (Invitrogen, CA) to release RNA. Next, the RNA was purified and isolated using the miRNeasy Mini Kit (Qiagen). The purified RNA was then quantified and assessed for purity with the Nanodrop ND-1000 system (Thermo Fisher Scientific, Wilmington, NC), ensuring OD260/280 ratios ≥1.7. RNA quality was assessed using an Agilent 2100 Bioanalyzer system with RNA 6000 Nano reagents (Agilent Technologies CA), where samples demonstrating RNA integrity numbers (RINs) ≥6 were deemed acceptable for downstream analysis. For further investigation of miRNA expression, 100 ng of total RNA underwent 3’-end dephosphorylation followed by Cy3-pCp labeling. The labeled RNAs were then hybridized to a Rat miRNA Microarray Kit (Rel21.0, Agilent Technologies) following the manufacturer’s standardized protocol.” 2.6 miRNA microarray Total miRNA was derived from five control and five model rat samples. Genome-wide miRNA expression profiling was performed using an Agilent Rat miRNA Microarray Kit (8×60K format, Agilent Technologies, Santa Clara, CA), which contains probes targeting all 758 rat miRNAs annotated in miRBase Release 21. Following hybridization, arrays were scanned at 3 μm resolution using an Agilent SureScan microarray scanner (G4900DA), with raw fluorescence intensities extracted using Agilent feature extraction software (v12.0.1). Data generation and normalization were performed using Agilent GeneSpring software (v.10.10). For miRNA expression data analysis, we utilized Python for standardization and log10 transformation, generating expression density curves for each sample. Principal component analysis (PCA) was subsequently conducted, where the data were standardized, the covariance matrix was calculated, and the eigenvalues were extracted. Finally, samples from the case and control groups were visualized in two-dimensional and three-dimensional space. Differential expression analysis was performed by log2 transformation (CLUSTER 3.0), which identified significantly altered miRNAs with absolute fold changes greater than 2 (|log₂FC| > 1). Hierarchical clustering was performed to establish the exosomal miRNA expression profile, and heatmaps were generated to compare the two groups. 2.7 Target prediction and enrichment information In this study, miRNA target genes were predicted using the miRanda algorithm, which assesses both sequence complementarity and the thermodynamic stability of miRNA–mRNA interactions. The prediction thresholds were set as a match score ≥150, free energy (ΔG) ≤ −30 kcal/mol, and strict 5′ seed region pairing. On the basis of the predicted targets, we subsequently conducted Gene Ontology (GO) analysis with a focus on biological processes and KEGG pathway enrichment analysis using the DAVID online tool (https://david.ncifcrf.gov/). 2.8 Validation by qRT‒PCR for selected miRNAs and mRNAs On the basis of target gene prediction, enrichment analysis, and literature screening, we selected exosomal miRNAs (miR-182 and miR-133b-3p) for experimental validation. Total RNA was extracted from cardiac exosomes (n=5 per group) and reverse-transcribed using miRNA-specific primers. Quantitative analysis was conducted using an ABI 7500 system with a SYBR Green qPCR Kit (ABclonal, RK21203) following the manufacturer’s instructions. The qPCR mixture was prepared to a final volume of 20 μL, comprising 2 μL of cDNA template combined with 10 μL of 2× SYBR Green PCR master mix, 0.4 μL each of 10 μM forward and reverse primers, and 7.2 μL of RNase-free water. The thermal cycling protocol comprised an initial denaturation step performed at 95 °C for 3 min, followed by 45 amplification cycles (5 sec at 95 °C for denaturation and 30 sec at 60 °C for combined annealing/extension). Technical triplicates were included for each sample, with miR-16 serving as the normalization control. In parallel, the expression levels of the predicted target mRNAs (Taok2, Aipl1, Gabra4, Iqgap1, and Rgs3) were quantified using the same RNA samples and normalizing to GAPDH expression. Relative expression levels were calculated using the 2^−ΔΔCt method. Diagnostic performance was evaluated through ROC curve analysis (GraphPad Prism) with AUC calculations to identify biomarkers with significant discriminatory power. 2.9 Statistical analysis Statistical analyses were conducted with PASW 20.0 software. Continuous variables were analyzed differently according to their distribution patterns: nonnormally distributed parameters are shown as median values with interquartile ranges (IQRs) and were compared using nonparametric tests, whereas normally distributed variables are described as the means ± SDs and were analyzed with t tests. The classified data are presented as percentages and were analyzed for between-group differences using chi-square tests or Fisher’s exact tests. The candidate miRNA and mRNA expression levels are presented as medians (IQRs), and differences between the model and control groups were evaluated with t tests. The sensitivity and specificity of exosomal miRNAs and mRNAs in disease diagnosis were evaluated via receiver operating characteristic (ROC) curves and their underlying areas (AUCs). ROC analysis and AUC comparisons were performed using SPSS. Moreover, Pearson correlation coefficients were used to analyze the relationships between miRNA and mRNA expression levels. The results with p<0.05 in two-tailed analyses were regarded as statistically significant. 3 Results 3.1 Dox affects the cardiac function of rats Anesthetized control and model group SD rats were scanned using a Mylab Seven ultrasound machine; the results are presented in Table 1 and Figure 1A. References indicate that an EF < 65% in echocardiographic assessment confirms successful modeling of chronic heart failure. As shown in Table 1, the EF value is significantly lower in the model group than in the control group, indicating successful model establishment. The echocardiograms and macro images of the heart are presented in Figure 1B. As shown in Figure 1C, compared with the control group, the model group presented markedly elevated serum levels of CK-MB, cTnI, and BNP, demonstrating that DOX induces substantial cardiotoxicity in rats. 3.2 Validation of the extracted exosomes TEM and Western blot analysis were performed on the exosomes extracted from the two groups of rats. As shown in Figure 2A, TEM revealed the presence of bilayer vesicular structures in both the control and model groups. Western blotting was employed to characterize exosomal markers, including positive markers (Alix, TSG101, CD63, and CD9) and the negative marker calnexin, using a molecular weight marker for reference. The results revealed bands for positive markers, whereas calnexin expression was not detected (Figure 2B). NTA revealed that the mean size of the exosomes obtained from the control group was 113.4 nm, whereas that of the exosomes from the model group was 130.3 nm (Figure 2C). In addition, we concluded that all the obtained vesicles displayed key characteristics of exosomes and were suitable for further experiments. 3.3 miRNA microarray performance Density distribution analysis of the miRNA expression data revealed that the expression distribution curves of all the samples largely overlapped, indicating that the overall expression patterns among the samples were similar and that the data quality was satisfactory (Figure 3A). Principal component analysis (PCA) was used to assess the consistency of the samples detected by the microarray(Figure 3B and C).The results demonstrated that the five samples within the same group were closely clustered in both two-dimensional and three-dimensional space, suggesting that these genes were representative across the samples, indicating good biological replicates. Exosomal miRNA profiles were analyzed using an Agilent Rat miRNA Microarray Kit. The array results revealed distinct miRNA regulation in the exosomes between the control group and the model group. Among the 467 exosomal miRNAs, 25 miRNAs (miR-122-5p, miR-124-3p, miR-125b-5p, miR-127-3p, miR-128-3p, miR-129-5p, miR-132-3p, miR-182, miR-183-5p, miR-200a-3p, miR-200b-3p, miR-212-5p, miR-219a-2-3p, miR-300-3p, miR-3553, miR-370-3p, miR-379-3p, miR-379-5p, miR-409b, miR-411-5p, miR-434-3p, miR-434-5p, miR-541-5p, miR-7a-5p, and miR-9a-5p) were upregulated, whereas one miRNA (miR-133b-3p) was downregulated(Figure 4D and E). 3.4 GO terms and KEGG pathway annotation of the miRNA targets On the basis of the differentially expressed miRNAs, potential target genes were predicted using the miRanda algorithm. GO enrichment analysis revealed that the predicted target genes were significantly enriched in several biological processes, including cell adhesion and structural maintenance (e.g., desmosome formation), cardiac electrophysiology and contraction (e.g., pacemaker conduction, calcium channels, and the troponin complex), and synaptic function (e.g., vesicle transport and synaptic plasticity) (Figure 4A). Subsequent KEGG pathway analysis revealed that the target genes were enriched mainly in metabolic and endocrine diseases (e.g., type 2 diabetes), cardiovascular diseases (e.g., cardiomyopathy), neural signaling, and MAPK/calcium signaling pathways (Figure 4B). 3.5 Candidate miRNAs and their target mRNAs associated with DCM This study aimed to identify differentially expressed miRNAs and their target mRNAs and to explore their potential value in disease diagnosis. First, qRT‒PCR analysis revealed that rno-miR-182 was significantly downregulated in the model group (P < 0.01), whereas rno-miR-133b-3p did not significantly change (Figure 5A). Further ROC curve analysis demonstrated that rno-miR-182 exhibited high diagnostic accuracy (AUC = 0.8533), suggesting its potential as a biomarker (Figure 5B). On the basis of miRNA target prediction, we subsequently examined the expression levels of five potential target mRNAs. Taok2, a predicted target of rno-miR-182, was significantly downregulated in the model group (P < 0.01), whereas Iqgap1 did not significantly change. Gabra4, a target of rno-miR-133b-3p, was significantly upregulated in the model group (P < 0.05), whereas Aipl1 and Rgs3 were not significantly different (Figure 5C). ROC analysis indicated that Taok2 (AUC = 0.8267) had good discriminatory ability, suggesting its potential value in disease diagnosis (Figure 5D). In summary, miR-182 may affect the MAPK pathway by targeting Taok2, and both exosomal miR-182 and Taok2 have diagnostic potential for doxorubicin-induced cardiotoxicity. 4 DISCUSSION Doxorubicin, the most commonly used anthracycline chemotherapeutic agent in clinical practice, has dose-dependent cardiotoxicity, which has become a major limiting factor for its therapeutic efficacy. Previous studies have shown that when the cumulative dose reaches 5–25 mg/kg, the mortality rate increases significantly to 10–38% and may further increase to 50% within two years after treatment [15] . This cardiotoxicity involves complex pathophysiological mechanisms, including oxidative stress [16] , mitochondrial dysfunction [17] , autophagy [18, 19] , and regulated cell death [20] . In recent years, the role of exosomes and their noncoding RNA cargo in cardiovascular diseases has attracted increasing attention. Our study revealed that myocardial-derived exosomal miR-182 is significantly upregulated in DCM, and bioinformatic analysis revealed that it may target the MAPK pathway-related gene Taok2, providing insight into novel mechanisms of DCM. Exosomes, which are lipid bilayer-enclosed cellular vesicles, can be actively secreted by various eukaryotic cells and participate in intercellular communication [21] . These vesicles are rich in bioactive molecules such as proteins and nucleic acids (including DNA, miRNAs, and lncRNAs), among which miRNAs have become a research focus owing to their stability and regulatory capacity. Numerous studies have demonstrated that exosomal miRNAs play crucial regulatory roles in various diseases. In oncology, expression profile analysis of miR-21, miR-451, and miR-636 in urinary exosomes from patients with prostate cancer revealed that their predictive accuracy for metastatic risk surpasses that of traditional prostate-specific antigen (PSA) testing [22] . Exosomal miR-144-3p derived from osteosarcoma can induce ferroptosis in tumor cells by targeting ZEB1, significantly suppressing tumor progression [23] . In cholangiocarcinoma-related exosomes, miR-182/183-5p enhances tumor stemness characteristics by the HPGD/PGE2 pathway [24] . In neurological diseases, the level of exosomal miR-124-3p derived from spinal motor neurons in patients with amyotrophic lateral sclerosis is positively correlated with disease severity [25] . Exosomal miR-137 delivered by microglia can attenuate cerebral ischemia‒reperfusion injury by inhibiting the Notch1 signaling pathway [26] . Recent studies have also revealed that the exosomal miRNA Novel-3 derived from foam cells participates in the progression of ischemic stroke by activating neuroinflammation and ferroptosis [27] . Studies of cardiovascular diseases have also confirmed the important regulatory role of exosomal miRNAs. Abnormal expression of plasma exosomal miR-342-3p in patients during the recovery phase of acute myocardial infarction is closely associated with impaired cardiac repair [28] [. In patients with atrial fibrillation, exosomal miR-124-3p promotes myocardial fibrosis by regulating AXIN1 [29] . In terms of therapy, various exosomal miRNAs exhibit significant cardioprotective effects. [30] By modulating hepatocyte growth factor and fibroblast growth factor β, miR-125a can significantly promote angiogenesis, whereas miR-25-3p exerts antiapoptotic effects by regulating the expression of the proapoptotic genes PTEN and FASL in cardiomyocytes [31] . However, studies on the role of myocardial-derived exosomal miRNAs in DCM remain very limited. Enrichment analysis of the target genes of the differentially expressed miRNAs revealed that these miRNAs may be involved in the pathogenesis of DCM through multiple mechanisms. GO analysis revealed that the associated target genes were significantly enriched in key biological processes, such as cardiac troponin complex formation and the regulation of myocardial contraction. KEGG pathway analysis revealed significant enrichment of target genes in the MAPK signaling pathway. Zhang et al. demonstrated that oxidative stress and cardiomyocyte apoptosis are among the core molecular mechanisms of DCM, with aberrant activation of the MAPK signaling pathway—especially p38 MAPK—playing a key regulatory role. Specific inhibition of p38 MAPK pathway activation has been identified as a potentially effective strategy for the prevention and treatment of DCM [32] . Recent studies have suggested that activation of the MAPK signaling pathway directly mediates DCM by promoting cardiomyocyte apoptosis, exacerbating oxidative stress, and inducing mitochondrial injury, whereas inhibition of this pathway alleviates toxicity [33] . On the basis of our findings, we speculate that miR-182 may regulate the activity of the MAPK signaling pathway by targeting Taok2, thereby playing a regulatory role in the development of DCM. This study revealed that exosomal miR-182 and Taok2 exhibited statistically significant differential expression in the model group, suggesting their critical role in DCM. Previous studies have shown that miR-182 is involved in the pathological processes of various diseases. In oncology, miR-182 influences the tumor microenvironment and metastatic progression by regulating signaling pathways such as the TGFβ/TLR4/NFκB and Wnt/β-catenin pathways [34, 35] . In neurological disorders, miR-182 contributes to neuronal injury by suppressing apelin, a neuroprotective molecule [36] . In the cardiovascular system, studies have confirmed that miR-182 promotes myocardial hypertrophy via NO-dependent activation of the Akt/mTORC1 pathway and targets PAPPA to inhibit vascular smooth muscle cell proliferation [37, 38] . However, although Taok2 has been implicated in neurodevelopmental disorders [39] , its role in DCM remains unclear. This study is the first to reveal the potential association between the miR-182/Taok2 regulatory axis and DCM, although its specific molecular mechanisms require further experimental validation. Although our study yielded important findings, several limitations remain. First, the sample size was relatively small, and further studies with larger cohorts are needed to increase the reliability of the conclusions. Second, although bioinformatics analysis revealed that miR-182 may target Taok2, this interaction has not yet been directly validated by methods such as dual-luciferase reporter assays. Finally, this study was based primarily on an animal model, and owing to biological differences between species, validation with clinical samples is needed to assess its translational relevance. In summary, this study conducted microarray expression profiling of exosomal miRNAs in DCM rats and identified 26 differentially expressed miRNAs (25 upregulated and 1 downregulated). Through bioinformatic analysis and qPCR validation, we confirmed that the level of exosomal miR-182 was clearly elevated in the DOX-treated group, which was accompanied by a corresponding reduction in the mRNA level of its target gene Taok2. ROC curve analysis indicated that exosomal miR-182 (targeting Taok2) may serve as a potential biomarker for DCM. These findings suggest that myocardial exosomal miR-182 may contribute to DOX-induced cardiotoxicity by modulating the Taok2/MAPK signaling pathway, offering new insights for clinical research. 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Table 1.Cardiac Function Parameters in Rats with Doxorubicin-Induced Heart \received DD MMMM YYYY \acceptedDD MMMM YYYY Control（n=3） Model（n=3） t P Ejection feaction% 73.96%±10.83 54.80%±10.55 4.32 <0.001 Fractional shortening % 38.74%±9.97 24.91%±6.3 3.87 <0.001 LVIDd cm 0.60±0.045 0.64±0.040 2.15 0.047 LVIDs cm 0.37±0.036 0.48±0.07 4.01 <0.001 Figure1. Doxorubicin-induced cardiac dysfunction in rats.A.Cardiac Function Parameters in Rats with Doxorubicin-Induced Heart Failure.B.Echocardiograms of Rats in Each Group.C.Determination of BNP, CK-MB, and cTnI in cardiac tissue from the two groups of rats.*: p Figure2. Characterization of exosomes in DCM model.A.Transmission electron microscopy results for the control and model groups of exosomes.B.Western blot detection of surface proteins in exosomes.C.Particle size of myocardial exosomes from rats in both the control and model groups, measured using a NanoSight instrument. Figure3. Comprehensive analysis of exosomal miRNA microarray in DCM.A.miRNA expression density across samples. B and C. Principal component analysis plot displaying sample clustering patterns.D.Volcano plot of differentially expressed exosomal miRNAs between the case and control groups.E.Heatmap and hierarchical cluster analysis of the exosomal miRNA microarray data from the case and control groups. Figure4. Functional enrichment analysis of differentially expressed exosomal miRNAs in DCM. A. Bar chart of the top 10 enriched genes according to GO enrichment analysis.B.Top 20 bubble charts from KEGG enrichment analysis top 20 bubble chart. Figure5. Differential expression and diagnostic potential of miRNAs and their target mRNAs in DCM. A.Relative expression levels of two candidate miRNAs (miR-182 and miR-133b-3p) between the model and control groups,measured by qRT-PCR.B. ROC curve analysis of the two differentially expressed miRNAs.C. Relative expression levels of five target mRNAs (Taok2, Aipl1, Gabra4, Iqgap1, and Rgs3) between the model and control groups.D. ROC curve analysis of the five target mRNAs.*: P < 0.05, **: P < 0.01 Supplementary Material File (figure.docx) Download 919.15 KB File (table.docx) Download 11.41 KB Information & Authors Information Version history V1 Version 1 07 August 2025 Peer review timeline Published Gene Version of Record 1 Jan 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cardio-oncology doxorubicin-inducedcardiomyopathy mir-182 myocardium-derived exosomal mirnas taok2 Authors Affiliations Cui Li 0009-0007-8701-573X Ningbo City First Hospital Department of Cardiology View all articles by this author Yudie Song Ningbo City First Hospital Department of Cardiology View all articles by this author Binbin Cao Ningbo City First Hospital Department of Cardiology View all articles by this author Jiahui Li Ningbo City First Hospital Department of Cardiology View all articles by this author Fan Xu Ningbo City First Hospital Department of Cardiology View all articles by this author Weiping Du Ningbo City First Hospital Department of Cardiology View all articles by this author Zhaoxia Zhang Ningbo City First Hospital Department of Cardiology View all articles by this author Xiaomin Chen Ningbo City First Hospital Department of Cardiology View all articles by this author Qinglin Yu Ningbo City First Hospital Department of Chinese Traditional Medicine View all articles by this author Jia Su 0000-0003-2163-2739 [email protected] Ningbo City First Hospital Department of Cardiology View all articles by this author Metrics & Citations Metrics Article Usage 150 views 98 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Cui Li, Yudie Song, Binbin Cao, et al. 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