Impact of pro-inflammatory monocyte subsets and their microRNAs regulation after an ischemia/reperfusion myocardial injury

Impact of pro-inflammatory monocyte subsets and their microRNAs regulation after an ischemia/reperfusion myocardial injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impact of pro-inflammatory monocyte subsets and their microRNAs regulation after an ischemia/reperfusion myocardial injury Elisa Bevilacqua, Débora Falcón, Isabel Galeano-Otero1, Francisco Morón-Civanto, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9448122/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Ischemia-reperfusion (I/R) injury in myocardial infarction with ST-segment elevation (STEMI) can lead to detrimental effects on the myocardium. Although primary percutaneous coronary intervention (PPCI) has significantly improved patient survival, some patients still develop adverse left ventricular remodeling, primarily due to interstitial fibrosis. The aim of this study was to investigate the role of pro-inflammatory cell populations and their released microRNAs (miRNAs) in regulating cardiac fibrosis and apoptosis in a rat model of I/R. Methods Flow cytometry was used to measure the levels of pro-inflammatory cell subsets. Pro-inflammatory neutrophils and monocytes were isolated from peripheral blood and subsequently used in an in vitro co-culture secretome assay to stimulate cardiac fibroblasts and cardiomyocytes. Microarray analysis of genes and miRNAs was performed on the two types of sorted pro-inflammatory monocytes 24h post-I/R to identify the inflammatory mediators responsible for pro-fibrotic and apoptotic genetic changes. Results Increased levels of neutrophils and monocytes, similar to those observed in humans, were detected following I/R, concomitant with the presence of M1 macrophages within rat cardiac tissue. The inflammatory secretome derived from these populations was found to induce pro-fibrotic and pro-apoptotic gene expression in cardiac fibroblasts and cardiomyocytes. Gene microarray analysis revealed significant differences in the transcriptome of rat monocyte types, consistent with findings in human mononuclear cells from STEMI patients post-revascularization. Moreover, miRNAs microarray analysis identified differences in the expression of miR-16, miR-27, miR-29, miR-30, and miR-194, which are associated with pro-apoptotic and fibrotic genes regulation, 24h after the I/R procedure. Furthermore, miRNAs mimics of some of these miRNAs changed the gene transcription in fibroblasts and cardiomyocytes. Conclusions Through combined co-culture analysis and miRNAs microarray screening, we were able to identify miRNAs as inflammatory mediators that modulate gene expression in myocardial and non-myocardial cells after I/R injury. These identified miRNAs could be used as therapeutic targets for reducing fibrosis and apoptosis after an ischemic event. I/R injury myocardial infarction Inflammatory monocytes miRNAs cardiac fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background An ischemic event resulting from coronary artery occlusion initiates a cascade of structural, functional, and metabolic disturbances that impair myocardial performance. Primary percutaneous coronary intervention (PPCI) remains the most effective treatment for limiting cardiac cell death and reducing the risk of adverse cardiac events following a severe ST-segment elevation myocardial infarction (STEMI) ( 1 ). However, despite timely PPCI, a substantial proportion of STEMI patients still develop adverse cardiac remodeling post-PPCI ( 2 ), involving structural and molecular changes that compromise cardiac performance and contribute to the progression toward heart failure (HF) ( 3 ). Among the various pathophysiological processes triggered by STEMI, fibrosis and apoptosis are key contributors to adverse remodeling following the ischemic event ( 4 ). Fibrosis results in excessive extracellular matrix deposition, impairing myocardial elasticity and function, while apoptosis contributes to the loss of cardiomyocytes, weakening the contractile capacity of the heart and exacerbating cardiac dysfunction. Peripheral blood-derived monocytes and neutrophils play a pivotal role in both cardiac injury and repair processes, facilitating wound healing through temporally distinct actions ( 4 ). Neutrophils are rapidly recruited to the infarcted myocardium, initiating a pro-inflammatory response, while monocytes infiltrate later and differentiate into macrophages that support tissue repair and promote resolution of inflammation ( 5 ). However, excessive or prolonged activation of these innate immune cells can exacerbate tissue damage, contributing to adverse remodeling and increasing the risk of HF and other cardiac events ( 6 ). Nevertheless, the exact mechanisms through which innate immunity impacts ventricular adverse remodeling and progression to HF remain incompletely understood. Monocytes represent a heterogeneous cell population classified into three subsets in humans, each characterized by specific phenotypic markers and functional roles during inflammation ( 7 ). In rats, monocytes immunodefined as CD43 low His48 high and CD43 high His48 low are thought to correspond to the classical CD14 ++ CD16 − and non-classical CD14 + CD16 ++ human monocytes subsets, respectively ( 8 , 9 ). Despite these proposed analogies, the precise functional roles of these rat monocyte subsets remain to be elucidated ( 10 ), highlighting the need for further phenotypic and functional characterization, especially in the context of inflammatory and ischemic conditions. Our previous studies demonstrated that inflammatory cell populations can produce a repertoire of pro-inflammatory mediators as chemokines, cytokines, microRNAs (miRNAs), as well as factors involved in the resolution of inflammation that could be used as prognostic markers ( 11 , 12 ). miRNAs belong to non-coding RNA family extensively involved in cardiovascular diseases prognosis and regulation ( 13 ). Growing evidence further supports the role of miRNAs not only as biomarkers of cardiovascular disease but also as key post-transcriptional regulators of gene expression implicated in cardiac remodeling ( 14 ). In this study, we investigated the contribution of ischemia/reperfusion (I/R) rats monocytes and neutrophils to adverse cardiac remodeling. Specifically, we examined their capacity to modulate the expression of fibrosis- and apoptosis-related genes in cardiomyocytes and fibroblasts. Furthermore, we analyzed the dysregulation of miRNAs in sorted rat monocytes and explored their potential regulatory roles in gene networks associated with fibrosis and apoptosis. Methods This study was conducted following the principles published by the declaration of Helsinki and its modification or similar ethical standards. The study was authorized by the local Ethics Committee on Human Research at the University Hospital “Virgen del Rocio” of Seville (Approvals no. 2013PI/096, 2018/352, 2021/03 and FIS-ISG-2024-01). Experiments with animals were performed in accordance with the recommendations of the Royal Decree 53/2013 in agreement to the Directive 2010/63/EU of the European Parliament and approved by the Animal Research Committee of the University of Seville (18/08/2020/097 and 22/07/2024/110). Study with patients All the subjects of this study were voluntary participants and signed an informed consent form. A cohort of 121 patients were prospectively recruited between March 11th 2016 and September 1st 2023. The participants were divided into two groups: 33 healthy volunteers (49 ± 12 years; 48.5% male sex) were persons who do not suffer arterial hypertension, dyslipidemia or diabetes mellitus type II, do not smoke and without diagnosed coronary disease, and 88 who were diagnosed with STEMI (58 ± 10 years; 84.1% male sex) and treated with a primary percutaneous coronary intervention (PPCI) through the right radial artery at the University Hospital “Virgen del Rocio” of Seville. Clinical and demographical information were collected at the admission in the hospital and during each follow-up visit, scheduled at 1 and 6 months after the hospital discharge. 51% completed the follow-up. The inclusion criteria were, age less than 75 years; patients with STEMI due to occlusion of the left descending artery with an epicardial blood flow TIMI (Thrombolysis in Myocardial Infarction) grade 0–1 in the initial angiogram treated with PPCI, with the onset of symptoms less than 12h before the angioplasty. The exclusion criteria were, ischemic heart disease history, a < 30 ml/min glomerular filtration rate, and a TIMI flow grade over 1. Rat cardiac surgery animal model Male healthy Wistar rats weighing 250 ± 50 g were anesthetized with intraperitoneal mixture of 50 mg/kg ketamine plus 8 mg/kg xylazine and maintained in 2% sevoflurane and oxygen described previously ( 14 ). A left thoracotomy was performed between the third and fourth rib, followed by a pericardiotomy. The left coronary artery was occluded with a 5 − 0 ProleneTM silk suture (Ethicon, Spain) and the knot was performed over a tube placed into the suture releasing the occlusion after 40 min to perform an ischemia/reperfusion model. The left coronary artery occlusion was confirmed by visual observation of cyanosis and ST-segment elevation by continuous ECG monitoring. The chest cavity was closed, and the air was expelled from the chest. Analgesia was induced with meloxicam (1 mg/kg) administered subcutaneously. Rats were left on a heating pad until fully conscious recovery; mortality during the interventions was 3%. For this study, we considered the following experimental groups: I/R, ischemia and reperfusion model with left coronary artery ligation during 40 min, and SHAM, where left thoracotomy was performed without touching the heart to remove the basal inflammation due to the intervention, and control rats. Blood samples were obtained at different time points (24h, 72h, 1 week, 4 weeks), while tissues (heart and spleen) were extracted at the time point of 72h, 1 and 4 weeks. Blood and tissue samples: extraction and preparation Human peripheral blood samples were collected into ethylenediaminetetraacetic acid (EDTA)-coated tubes (BD Vacutainer® K2E; BD Biosciences, USA) and extracted before revascularization (STEMI 0h) and after culprit vessel opening (STEMI 6h, 1 and 6 months). 100 µl of blood was incubated during 30 min at room temperature (RT) with CD11b, CD14 and CD16 and CD66b antibodies (APC Mouse Anti-Human CD11b/Mac-1 clone ICRF; FITC Mouse Anti-Human CD14 clone M5E2; PE Mouse Anti-Human CD16 clone 3G8; PerCP-Cy™5.5 Mouse Anti-Human CD66b clone G10F5, respectively; BD Pharmingen, USA). Then, red blood cells were lysed with BD FACS™ Lysing Solution 10X Concentrate (BD Biosciences, USA) following manufacture instructions and washed with phosphate buffered saline (PBS 1X; GIBCO, USA) and centrifuged. The pellet was resuspended in 300 µl of PBS 1X and analyzed by flow cytometry. Human peripheral blood mononuclear cells (PBMCs) were extracted as previously described ( 12 ). Briefly, 3 ml of EDTA peripheral blood was diluted with PBS 1x 1:1 and deposited above Ficoll (Lymphosep- Lymphocyte Separation Medium, MP Biomedical, USA) following manufacture instructions. After centrifugation at RT for 20 min at 1600 rpm without brake, PBMCs were recollected from the above phase and washed with PBS 1X. Supernatant was removed, and the pellet was frozen and keep to -20ºC for RNA extraction. Rats red blood cells were lysed with distilled water during 1 min 30 sec and the lysis was stopped with 1.8% NaCl solution. Spleen homogenate was obtained by half of the organ shredding and red blood cells were lysed with distilled water during 1 min. Cells were incubated with 1 µl of His48, CD11b and CD43 antibodies (Mouse Anti- Rat Granulocyte Marker His48 FITC, Invitrogen, USA; Mouse Anti- Rat CD11b APC, BD Pharmingen, USA; Mouse Anti- Rat CD43 Alexa Fluor 594, R&D systems, USA). Rat hearts were directly included in Tissue-Tek® O.C.T. Compound (Sakura Finetek, USA) and frozen for immunofluorescence or fixed with formalin and embedded in paraffine following the dehydration process for trichrome Masson’ staining. Flow cytometry measurements Human blood samples were acquired in a Canto II flow cytometer (BD Biosciences, USA), while the rat samples were analyzed at LRS II Fortessa flow cytometer (BD Biosciences, USA) and sorted at MoFlo Astrios FACS (Beckman Coulter, USA). Sample acquisition and analysis was performed with the FACS-Diva software 8.0 (BD Biosciences, USA). The inflammatory human cell populations analyzed were, neutrophils (CD16 ++ CD66b + ), eosinophils (CD16 + CD66b − ) and monocyte subsets. Monocyte subgroups were classified as CD14 ++ /CD16– (classical), CD14 ++ /CD16 + (intermediate), and CD14 + /CD16 ++ (non-classical). The inflammatory rat cell populations analyzed were, neutrophils (FSC/SSC, CD11b + ), and the two monocyte subsets: CD11b + CD43 high His48 low (non-classical) and CD11b + CD43 low His48 high (classical) monocytes. Immunofluorescence and histology staining For trichrome Masson staining, hearts were fixed in formalin, dehydrated, embedded in paraffin and sectioned at 6 µm using a microtome. Tissue sections were then stained according to manufacturer's instructions to visualize fibrosis. Images were acquired using an Olympus BX-61 direct microscope and analyzed with a proper pipeline to detect fibrosis at CellProfiler software. For immunofluorescence experiments, hearts were sliced at the cryostat with a width section of 6 µm and then were fixed with formalin for 30 min. Slices were washed with PBS 1X and permeabilized with PBS 1X- 0.3% Triton (Triton X100, Sigma) during 15 min. Subsequently, to block unspecific binding of the antibodies, sections were incubated with PBS 1X- 0.1% Triton- 10% Goat serum (Gibco, USA)- 1% BSA (Bovine Serum Albumin, A2153-100G, Sigma, USA) for 1h. Tissues were incubated with appropriate diluted antibodies (CD163, ED2 clone 1:100; CD68, ED1 clone 1:100, Biorad, USA) in blocking solution in a humidified chamber overnight at 4°C. Slices were washed and then incubated with secondary antibodies (Alexa Fluor 488 goat anti-mouse, Alexa Fluor 594 goat anti-mouse, 1:400, Thermo Fisher Scientific, USA) in blocking solution for 3h at RT in the dark. Finally, nuclei were stained with DAPI (4',6-diamidin-2-fenilindolo, Sigma, USA) and coverslip were mounted with a drop of mounting medium DAKO Fluorescence Mounting Medium (Dako, USA). Isolation of adult cardiac cells Cardiac populations, enriched in fibroblasts and cardiomyocytes, were isolated using a standard enzymatic digestion (Collagenase type II, 251 IU/mL; Worthington Biochemical, USA) as described previously ( 15 ). Briefly, hearts were removed and perfused on a Langendorff perfusion device. After perfusion, hearts were left in enzyme solution supplemented with 2 g/L BSA. Isolated cells were then filtered, centrifuged and suspended in Tyrode solution containing (in mM): 130 NaCl, 1 CaCl 2 , 0.5 MgCl 2 , 5.4 KCl, 22 glucose, 25 HEPES, 0.4 NaH 2 PO 4 , 5 NaHCO 3 ; pH was adjusted to 7.4 with NaOH. Cardiac populations were obtained with differential centrifugation (200 rpm for 1 min for cardiomyocytes, 1500 rpm for 5 min for fibroblasts). Cells were cultured on laminin (L2020, Sigma, USA) pretreated plates in control solution containing RPMI 1640 medium (Corning, USA) supplemented with 8% fetal bovine serum (FBS; Gibco, USA), 10% Streptomycin-Penicillin (10.000 units, Capricorn Scientific, USA) and 10% L- glutamine (200mM, Sigma, USA) at 37ºC. Granulocytes secretome medium preparation and cardiac cells stimulation Granulocytes obtained from peripheral blood of SHAM and I/R rats, 72h after the intervention, were used to stimulate myocardial populations. Neutrophils and monocytes were isolated with 10% dextran (Sigma, USA) in a proportion of 2:15. After 30 min of decantation, supernatant was collected and deposited above Ficoll (Lymphosep- Lymphocyte Separation Medium, MP Biomedical, USA). Monocytes were obtained from the above Ficoll phase, while neutrophils were collected from the pellet. The two populations were cultured with RPMI 1640 medium (Corning, USA) supplemented with B27 (Corning, USA), 10% Streptomycin-Penicillin (10.000 units, Capricorn Scientific, USA) and 10% L- glutamine (200mM, Sigma, USA) for 24h. Then, supernatant was collected and added in proportion 1:1 to fibroblasts and cardiomyocytes obtained before. Cells were cultured with the mixed medium containing 4% of FBS. 24h after the treatment, total RNA from fibroblasts and cardiomyocytes was collected. Neonatal cardiac cell culture and miRNA transfection For cell transfection analysis we used neonatal rat ventricle myocytes (NRVMs) and cardiac fibroblasts. They were isolated from hearts of 1–3 days old Wistar rats using trypsin as described previously ( 16 ). Following trypsinization, fibroblasts were separated from NRVMs based on their differential adhesion properties. Cardiac fibroblasts adhered to the culture dishes within 1h, while NRVMs exhibited poor attachment to the coated surface during this short period. After the 1h incubation, the supernatant containing the unattached cardiomyocytes was collected, and the fibroblasts remained attached to the plate. NRVMs were cultured in DMEM/M199 (4:1) (Gibco, USA) supplemented with 10% horse serum, 5% FBS, 1% glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin, while cardiac fibroblasts were grown in DMEM supplemented with 10% FBS, 1% glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. NRVMs and cardiac fibroblasts were transfected with miRNA mimics at 70% confluence, 48h post-isolation, following the manufacturer's instructions using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, USA). The cells were incubated for an additional 48h before subsequent experiments. Briefly, 6 µl of Lipofectamine was diluted in 250 µl of Opti-MEM® Medium (Gibco, USA), then 4,5 µl of miR-16 mimic, miR-27a mimic or miR-30b mimic (10 µM) (Thermo Fisher Scientific, USA) was diluted in 250 µl of Opti-MEM® Medium. The solutions were mixed in a 1:1 ratio and incubated for 10 min at RT. Then, mix was added to cells. Ischemia/reperfusion (I/R) was performed 24h post-transfection using a simulated ischemic solution (in mM): 142 NaCl, 3.6 KCl, 1.2 MgCl 2 , 1.8 CaCl 2 , 5 NaHCO 3 , 20 Hepes, 20 Lactate-Na, 20 sucrose; pH 6.22, in a hypoxia chamber (1% O 2 and 5% CO 2 ), for 3h. Reperfusion/reoxygenation started when the ischemic solution was removed and cells were placed in an incubator at 21% O 2 and 5% CO 2 in DMEM/M199 (4:1) supplemented with 0.4% horse serum, 0.07% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. MiRNAs and genes Array Arrays were performed from sorted monocytes of SHAM and I/R rats 24h after the intervention. Total RNA was extracted with miRNAeasy mini kit (Qiagen, Germany) according to the manufacturer’s instructions. The total cDNA was labeled using the FlashTag® Biotin HSR labeling Kit (Thermo Fisher Scientific, USA). GeneChip® miRNA 4.0 arrays (Thermo Fisher Scientific, USA) were used to analyze miRNAs, while Clariom S Assay Rat (Thermo Fisher Scientific, USA) was used to study gene expression. Washing, staining (GeneChip® Fluidics Station 450, Thermo Fisher Scientific, USA), and scanning (GeneChip® Scanner 3000, Thermo Fisher Scientific, USA) were done following manufacturer’s protocol. Briefly, importing CEL file, the analysis of miRNA level RMA+DABG-All and exporting the results were done using Transcriptome Analysis Console (TAC) 4.0 software (Thermo Fisher Scientific, USA). A comparative analysis between SHAM rats and I/R rats was carried out using fold-change of over ± 1.5 with a p-value < 0.05. In silico analysis of miRNAs regulating apoptosis and fibrosis related genes was performed with TAC 4.0 software. The different expressed miRNAs were studied with miRDB ( www.miRDB.com ) and TargetScan ( www.targetscan.org ) to evaluate possible target genes. Later, the target genes were filtered with GeneVenn ( www.genevenn.sourceforge.net ). Target genes implicated in different biological pathways were studied with Panther Gene ( www.pantherdb.org/ ). Enrichment analysis for genes was performed firstly with Gene Ontology ( www.geneontology.org ) and then with GSEA 4.3.2 (Gene Set Enrichment Analysis), using Biological Processes C5 library 6.2 version. RNA extraction and RT-qPCR For miRNA analysis, total RNA was extracted with miRNAeasy mini kit (Qiagen, Germany) according to the manufacturer’s instructions. To perform RT-qPCR, RNA was retrotranscribed to cDNA with miRCURY LNA RT Kit (Qiagen, Germany) for miRNAs detection. PCR mix for miRNAs included miRCURY LNA SYBR Green PCR kit (Qiagen, Germany) and hsa-miR-16-5p, hsa-miR-18a-5p, hsa-miR-27a-3p, hsa-miR-29b-3p, hsa-miR-30b-5p, hsa-miR-30e-5p, hsa-miR-125a-5p, hsa-miR-194-5p, hsa-miR-222-3p miRCURY LNA miRNA PCR Assay. Data analysis was made with QuantStudio™ Real-Time PCR Software (Thermo Fisher Scientific, USA). Fold change quantification was calculated using the comparative cycle threshold (CT) method, using 18S and U6 as endogenous control. Prime PCR was used as described previously ( 14 ). Briefly, the retrotranscription was performed according to the manufacture instruction with iScript™ Advanced cDNA Synthesis Kit (1725037, Biorad, USA). Apoptosis and survival Tier 1 H384 384 (10040258, Biorad, USA) was performed with SsoAdvanced Universal SYBR Green Supermix (1725270 EDU, Biorad, USA). The studied genes are listed in Supplemental Table 1. For gene analysis, RNA from patients’ PBMCs were extracted with miRVana (AM1560, Thermo Fisher Scientific, USA), while for cardiac populations NucleoSpin RNA (740955.50.00, Macherey-Nagel, USA) was used. Retrotranscription from 1 µg of mRNAs was performed with iScript cDNA Synthesis Kit (1708891, Biorad, USA) for all the samples and cDNA was diluted 1:10. qPCR was performed in a FrameStar 384 Well PCR Plate (4titude, BIOKé, Leiden, the Netherlands). PCR mix included SYBR Green reactive (iTaq™ Universal SYBR Green Supermix; Biorad, USA) and specific oligos for each gene, listed in Supplemental Table 2. RT-qPCR were performed on the Applied Biosystems Viia7 Real-Time PCR System (Thermo Fisher Scientific, USA). The thermal cycling conditions were as follows: 95°C for 20 sec followed by 45 cycles of 95°C for 1 sec and 60°C for 20 sec. Expression data were calculated like fold change obtained with the comparative cycle threshold CT (ΔΔCT) method, using 18S as endogenous control. Statistical Analysis Statistical analysis were performed with GraphPad (GraphPad Software, USA). Results are presented as mean and SD. Shapiro-Wilk was used as normality test. To compare normal data of 2 or more groups the t-student or the ordinary one-way ANOVA were used respectively, while for non-normal distributed data we used Mann-Whitney and Kurskall-Wallis test, respectively. Statistical significant differences were considered when p < 0.05. Results Inflammatory cells subsets dynamics in STEMI patients and in I/R rats . In order to study the level of inflammatory cells in humans after an ischemic insult, we recruited 121 participants, healthy volunteers without diagnosed coronary disease and 88 who were diagnosed with a ST-segment elevation myocardial infarction (STEMI) and treated with a Primary percutaneous coronary intervention (PPCI). STEMI patients showed an accused neutrophilia, before (0h), 6h after the revascularization and 1 month later the ischemic event, during the follow-up, compared to the control group (Fig. 1 A). Levels of neutrophils decreased 6 months after PPCI, approaching the levels analyzed in the control group. STEMI patients also showed significantly increased levels of classical monocytes at 6h, (Fig. 1 B), of intermediate monocytes at 0h and 6h (Fig. 1 C) and non-classical monocytes 1 month after PPCI (Fig. 1 D) compared to the control group. To complement the clinical findings, we evaluated inflammatory cell changes in a rat model of ischemia/reperfusion (I/R). We quantified neutrophils and monocytes levels in peripheral blood, from SHAM, I/R and rats without any intervention (Supplemental Fig. 1A-C), at different time points, 72h, 1 and 4 weeks after surgery procedures. As shown in Fig. 1 E-G, neutrophil levels augmented 72 h after the intervention in I/R rats, compared to SHAM operated rats, while 1 week after the operation this increase was not significant (Fig. 1 E). The analysis of monocytes sub-population indicates that I/R rats showed a marked increase in the level of CD43 high His48 low and CD43 low His48 high , especially 1 week post-surgery (Fig. 1 F, 1 G). By 4 weeks, I/R rats showed a significant reduction in monocyte and neutrophils levels, suggesting resolution of the inflammatory phase. To assess the contribution of extramedullary hematopoiesis after the myocardial infarction, we analyzed immune cell populations in the spleen of I/R rats. We observed no significant change in neutrophil levels in I/R rats, except for a significant reduction at 72h. (Supplemental Fig. 1D). Regarding the two monocytes subsets, they remained unchanged in I/R rats across all time points (Supplemental Fig. 1E, 1F). Collectively, these data confirm that the inflammatory response following an ischemic event in rats involves dynamic alterations in circulating and/or splenic neutrophils and monocytes, similar to STEMI patients. Cardiac macrophage infiltration after ischemic injury Peripheral blood monocytes infiltrate and differentiate into phagocytic macrophages in the heart after an ischemic event. Considering monocyte levels, we examined macrophage heart infiltration after I/R injury. Proinflammatory M1 macrophages, identified by CD68 + expression, were absent in SHAM hearts but were detected in I/R rats (Fig. 1 H-J). These cells were localized specially within the infarcted area at 72h (Fig. 1 H) and 1 week (Fig. 1 I) after the procedure but were no longer detected at 4 weeks (Fig. 1 J). By contrast, the no-inflammatory CD163 + macrophages, were evenly detected in all the experimental groups, exhibiting a homogenous distribution (Supplemental Fig. 1G, 1H). These data suggest that monocytes recruited to the injured heart differentiate into M1/CD68 + macrophages in the early phase at 72h and 1-week post-intervention, while M2/CD163 + macrophage levels remain relatively unchanged. Monocyte subtypes transcriptome is enriched in genes participating in inflammation and pro-fibrotic pathways. Next, we sought to examine the transcriptome changes of monocytes subsets after I/R, performing genes profiling on sorted monocytes (CD43 high His48 low and CD43 low His48 high ) from SHAM and I/R rats 24h post-intervention to analyze inflammatory genes in an early phase (Fig. 2 ). Principal Component Analysis (PCA) of the global transcriptome revealed pronounced segregation and well-defined clustering of the analyzed samples driven by both monocyte subtypes and experimental conditions (Fig. 2 A). The analysis identified 304 differently expressed genes in CD43 high His48 low monocytes, including 129 upregulated and 175 downregulated, while in CD43 low His48 high monocytes, 184 genes were differently expressed, with 68 upregulated and 116 downregulated, compared with analogous populations in SHAM operated rats (Fig. 2 B). Volcano plots illustrated these differentially expressed genes in both monocyte subsets, highlighting genes with high fold-changes and statistical significance (Fig. 2 C, 2 D). In addition, heatmap analysis indicated that gene expression profiles clustered reliably by cell type and condition (Supplemental Fig. 2A, 2B). To further understand the biological roles of the differentially expressed genes in activated monocyte subsets after I/R, we performed functional enrichment analysis using PANTHER classification system. The analysis revealed that both CD43 high His48 low and CD43 low His48 high monocytes were significantly enriched in pathways related to TGF- beta signaling and inflammation mediated by chemokine and cytokine signaling pathways. CD43 high His48 low monocytes also showed enrichment in angiogenesis, FGF and interleukin signaling pathways, among others (Fig. 2 E, 2 F). Furthermore, Gene Ontology (GO) enrichment analysis indicated that leukocyte regulation and extracellular matrix organization were enriched in CD43 high His48 low monocytes, both of which are closely linked to inflammatory responses (Supplemental Fig. 2C). In CD43 low His48 high , the analysis highlighted GO terms associated with the extrinsic apoptosis signaling pathway and chemokine production (Supplemental Fig. 2D). Finally, the GSEA (Gene Set Enrichment Analysis) further confirmed the existence of distinct functional gene expression profiles between the two monocyte subsets following I/R (Fig. 2 G, 2 H), supporting the hypothesis that each subset may play a specialized role in the post-ischemic inflammatory response. The secretome of immune cells isolated from I/R rats induces changes in fibrotic and apoptotic gene expression. Given the observed changes in neutrophils and monocytes in vivo in I/R rat models, we hypothesized that the secretome of these activated immune cells could directly modulate cardiac gene expression. Therefore, we exposed healthy rat cardiac fibroblasts and cardiomyocytes to a conditioned media collected from activated neutrophils and monocytes isolated from I/R rats (Fig. 3 A). Taking in consideration the transcriptomics results we focused on the potential role of these immune cell secretomes in modulating fibrotic and apoptotic processes after the ischemic injury. First, we confirmed that I/R induces cardiac fibrosis as soon as 1 week after the intervention, as assessed by Masson’s trichrome staining (Fig. 3 B, 3 C). Second, we analyzed the expression of fibrotic genes Col1 , Col3 and Tgfβ1 and apoptotic genes Apaf1, Bcl2 and Cycs in cardiomyocytes and fibroblast treated with immune cells secretome. Figure 3 D shows that the stimulation of cardiomyocytes with conditioned medium from monocytes isolated from I/R rats significantly increased Col1 and Col3 mRNA expression compared to SHAM controls, while neutrophil secretome had no significant effect on these gene expression. By contrast, only Tgfβ1 expression was significantly increased in cardiac fibroblasts stimulated with I/R neutrophils conditioned medium whereas monocytes secretome had no effect in fibrotic genes expression. Moreover, Fig. 3 E shows that the stimulation of cardiomyocytes with I/R monocytes conditioned medium trended to increase Cycs and Bcl2 expression. In contrast, exposure to I/R neutrophil conditioned medium led to a significant reduction in Apaf1 gene level, without affecting Bcl2 and Cycs expressions. In fibroblasts, I/R neutrophils supernatant significantly upregulated both Apaf1 and Bcl2 , whereas I/R monocytes conditioned medium do not affect their expression (Fig. 3 E). These findings further validate the distinct and cell-type-specific influence of monocyte and neutrophil secretomes on fibrotic and apoptotic gene regulation in cardiac cells. To assess whether these findings can be confirmed in patient samples, we analyzed the expression of fibrotic and apoptotic genes in PBMCs from STEMI patients. Samples collected before (0h) and 6–12h after revascularization were used to evaluate gene expression using a custom 75-gene Prime qPCR array. As shown in Supplemental Fig. 3A, PBMCs collected before revascularization (0h) exhibited a balanced pattern of up- and downregulated genes. However, 6–12h post-revascularization, we observed a predominant upregulation of gene expression (52 out of 75 assessed genes, Supplemental Fig. 3B). Subsequently, using RT-qPCR we confirmed that AKT2 , HMOX1 , MYC and NFKB1 were significantly upregulated in STEMI patients after the revascularization compared to controls, all of which are associated with apoptotic and fibrotic processes (Supplemental Fig. 3C). These results support the preclinical data and demonstrate that myocardial infarction induces widespread dysregulation of inflammation, fibrosis, and apoptosis-related genes in both animal model and human patients. miRNAs overexpressed in inflammatory monocytes modify fibrosis and apoptosis-related genes expression. Considering the previous results, we hypothesized that miRNAs expressed by the inflammatory monocytes could play a key role mediating the observed changes in gene expression. Therefore, we performed a miRNAs array analysis in sorted peripheral blood monocytes (CD43 high His48 low and CD43 low His48 high ) from I/R rats. PCA analysis showed clear separation and clustering of the samples (Fig. 4 A). We found 935 differently expressed miRNAs in CD43 high His48 low and 669 miRNAs in CD43 low His48 high (Fig. 4 B). Volcano plots and heatmaps illustrated these miRNAs expression profiles in both monocyte subsets from I/R rats compared to SHAM (Fig. 4 C, 4 D, Supplemental Fig. 4A). Using the datasets, we performed an in-silico analysis to determine which miRNAs are predicted to modulate previously characterized fibrotic genes ( Col1 , Col3 and Tgfβ1 ) and apoptotic genes ( Apaf1, Bcl2 and Cycs ) in I/R rats. (Fig. 4 E, Supplemental Fig. 4B). The analysis revealed 9 common miRNAs: miR-16-5p, miR-18a-5p, miR-27a-3p, miR-29b-3p, miR-30b-5p, miR-30e-5p, miR-125a-5p, miR-194-5p and miR-222-3p, in both types of monocytes. Subsequent validation by RT-qPCR confirmed that miR-16-5p, miR-27a-3p, miR-29b-3p, miR-30b-5p, miR-30e-5p, miR-194-5p were significantly upregulated in CD43 high His48 low monocytes from I/R compared to SHAM rats, but not miR-18a-5p, miR-125a-5p, and miR-222-3p (Fig. 5 ). Conversely, the level of these miRNAs did not change in CD43 low His48 high monocytes except for miR-27a-3p (Supplemental Fig. 5). To further characterize the regulatory mechanisms mediated by miRNAs, we selected miR-16-5p, miR-27a-3p and miR-30b-5p to explore their impact on the expression of fibrotic and apoptotic genes in cardiac fibroblast and cardiomyocytes. Rat neonatal fibroblasts and ventricular myocytes (NRVM) were transfected with mimics of these miRNAs under control conditions and following simulated I/R. As shown in Fig. 6 and Supplemental Fig. 6, I/R significantly increased Col1 and Col3 mRNA expression in both NRVM and fibroblasts. Interestingly, miR-16-5p and miR-30b-5p mimics significantly downregulated Col1 and Col3 expression in both cell types (Fig. 6 A), whereas miR-27a-3p mimic had no significant effect in NRVM (Supplemental Fig. 6). Notably, miR-30b-5p mimic upregulated TGFB1 expression in NRVM. We next examined the expression of apoptosis-related genes Apaf1 , Bcl2 , and Cycs under similar experimental conditions (Fig. 6 B and Supplemental Fig. 6). I/R stimuli led to a slightly increase of Apaf1 and decrease of Bcl2 in NRVMs and fibroblast, while their transfections with miR-16-5p mimic (Fig. 6 B) prevented I/R-induced Apaf1 and Bcl2 induced dysregulation in NRVM and/or fibroblasts. Conversely, miR-30b-5p mimic selectively downregulated Bcl2 in fibroblasts but did not affect Apaf1 expression in either cell type. Moreover, miR-27a mimic prevented I/R-induced Apaf1 upregulation and Bcl2 downregulation in either cell type (Supplemental Fig. 6). On the other hand, none of the tested miRNA mimics significantly modified Cycs gene expression in either fibroblasts or NRVM (Fig. 6 B; Supplemental Fig. 6). These findings indicate that miR-16-5p, miR-27a-3p, and miR-30b-5p distinctly regulate the expression of Bcl2 and Apaf1 in cardiac cells, thereby influencing the apoptotic processes initiated by I/R injury. In summary, our data demonstrate that miRNAs overexpressed in CD43 high His48 low monocytes from I/R rats, modulate the expression of fibrosis- and apoptosis-related genes in cardiac fibroblasts and cardiomyocytes. Discussion The inflammatory response following myocardial infarction plays a pivotal role in adverse cardiac remodeling with the infiltration and activation of neutrophils and monocytes populations driving fibrosis and cardiomyocyte apoptosis. However, the functional heterogeneity of monocyte subsets and the specific molecular mechanisms they regulate in this context, still require further comprehensive studies. Our work aimed to understand these functional and molecular diversity by isolating two types of rat monocytes after an I/R heart injury. We determined the differentially regulated gene expression profiles of these cell subtypes and analyzed their miRNAs as regulatory intermediates, implicated in inflammatory cardiac remodeling. The monocytosis in humans after an acute myocardial infarction (AMI) has been studied as adverse remodeling marker in some human data sets, as revascularized STEMI patients ( 12 ). In this study we found significantly high numbers of neutrophils and monocyte subsets in STEMI patients after a PPCI, consistent with our previous study ( 12 ). Furthermore, the analysis of pro-inflammatory genes expressed by PBMCs from STEMI patients, before and after the PPCI by prime-PCR showed that the expression of several genes changed as early as 6h to 12h after the revascularization during the inflammatory state. Pro-apoptotic genes as MYC and anti-apoptotic genes as AKT2, HMOX1 and NFKB1 were significantly upregulated in STEMI patients after PPCI. These increase suggest a molecular response toward cellular survival and stress adaptation in PBMCs. Actually, the induction of the AKT2 and NFKB1 pathways could reflects an enhanced anti-apoptotic signaling environment. These results align with a recent single-cell transcriptomic study, which demonstrated a systemic activation of the NF-KB pathway in some monocytes cell clusters following a STEMI in patients ( 17 ). Concomitantly, the increased expression of HMOX1 points to a cytoprotective mechanism against the acute oxidative stress secondary to cardiac damage, possibly modifying myocardial fibroblast phenotype as indicated previously ( 18 ), while the elevation of MYC transcripts may indicate cellular activation and metabolic reprogramming in response to systemic inflammatory state after the injury ( 19 ). Overall, these data indicate that PBMCs from STEMI patients undergo a rapid and coordinated transcriptional shift after PPCI, engaging both pro‑survival and stress‑response programs. After I/R injury, it is known that monocytes and neutrophils are the first immune cells to infiltrate and 24-48h after the insult, monocytes subsets are the most abundant cell types in the myocardium ( 20 , 21 ). Herein and using an I/R rat model we confirm that these monocytes-macrophages inflammatory populations persist for the first 72h-1 week after injury and subsequently decrease 4 weeks later, consistent with previous findings ( 22 ). Rat inflammatory cells are presumed to share similar properties with mice and human monocytes; however, these populations are partially studied in rat and remain poorly characterized. Recently, new membrane-markers of rat peripheral blood inflammatory cells were recently described allowing their detection by FACs ( 8 ). To address this gap, we performed a microarray to characterize gene transcription of these monocyte subsets following an I/R damage. The transcriptomic analysis revealed significant gene dysregulation in both populations, mainly associated with cardiac remodeling signaling pathway, including inflammation, fibrosis, and apoptosis. These findings are consistent with the well-established effect of the ischemic injury and inflammatory cell infiltration in the heart, which trigger extensive apoptosis and fibrosis replacement of dead cells ( 19 ). The plasticity of monocytes and macrophages is known to allow dynamic changes in their phenotype in response to microenvironmental signals ( 4 , 23 , 24 ). Functional differences among monocyte and macrophages subpopulations have been related to their inflammatory profiles and responsiveness to pro-inflammatory stimuli ( 25 ), which significantly may influence the immune regulation in ischemic heart diseases ( 26 ). Accordantly, our transcriptomics analysis showed differences between the two subtypes of monocytes CD43 high His48 low and in CD43 low His48 high after I/R stimulation, with a high percentage of variance reflecting extensive differential gene expression in each subset. Along the same line of research, a tissue single-cell RNA-seq study described that monocytes and macrophages are more diverse than expected ( 27 ) and their diversity in blood could be associated to cardiovascular risk factors such as smoking and hyperlipidemia ( 28 ). In our dataset, rat monocytes transcriptomic analysis showed well-differentiated cell populations after I/R damage, reflecting well-defined cell populations despite it is not a single-cell RNA-seq analysis. Moreover, monocytes have dynamic transcriptomes in thrombotic disease ( 29 ) and after myocardial infarction ( 30 ). Therefore, while single-cell sequencing techniques offer unprecedent insights and high-resolution into complex biological systems ( 31 ), complementary functional studies are still essential to elucidate these dynamic processes. Hence, we used the secretome of monocytes and neutrophils collected from I/R rats as stimuli for cardiac cells isolated form control rats to mimic immune cell communication with cardiomyocytes and fibroblasts, and to analyze the functional impact of immune cells on I/R-evoked adverse cardiac remodeling. Our findings suggest that neutrophil -derived factors, rather than monocytes, are more likely modulators of apoptotic gene expression while monocytes secretome influences fibrosis, consistent with previous results ( 21 ). Accordantly, others studies demonstrated that these pro-inflammatory cell populations are able to change fibroblast and cardiomyocyte gene expression by chemokines production ( 32 ), or other inflammatory cells derived molecules including non-coding RNAs ( 33 ). Here, we focused on the role of miRNAs, since our previous studies have demonstrated that miRNAs are rapidly dysregulated after PPCI in STEMI patients serum ( 11 ), in PBMCs ( 12 ), and in I/R rats serum and cardiac tissue ( 13 ). However, their specific role in monocytes after I/R has not been previously explored. Interestingly, we identify several dysregulated miRNAs expressed in CD43 high His48 low monocytes compared with CD43 low His48 high cells, suggesting their differential sensibility to I/R. The in-silico analysis identified a repertoire of miRNAs, targeting key genes implicated in fibrosis and apoptosis. Among the identified and validated miRNAs, miR-16-5p, miR-27a-3p, and miR-30b-5p are known to be key players in cardiovascular physiology, including fibrosis and apoptosis ( 34 – 36 ). Interestingly, the incubation of neonatal fibroblasts and cardiomyocytes with mimics of these miRNAs efficiently changed expression levels of fibrotic and apoptotic genes. Specifically, miR-16-5p mimic diminished the level of collagen genes both in cardiomyocytes and fibroblast consistent with its reported anti‑fibrotic effects ( 37 ). Moreover, miR-16-5p likely balances the expression of pro- and anti- apoptotic genes by increasing BCL2 expression, widely known for its anti-apoptotic action ( 38 ), and decreasing APAF1 expression, which has a recognized role in initiating apoptosis ( 39 ). Notably, this miRNA also predicts the adverse cardiac remodeling in STEMI patients ( 12 ). Concordantly with our results, the miR-30 family, including miR-30b-5p, is known for its anti-fibrotic properties, directly suppressing the expression of extracellular matrix components like collagens, alongside with its impact on apoptotic processes post-myocardial infarction ( 40 ). Finally, despite that miR-27a-3p exerts context-dependent effects on both fibrosis and apoptosis ( 41 , 42 ), we observed an anti-apoptotic effect of this miRNA in neonatal cardiomyocytes through inhibition of APAF1 . Altogether, these roles underscore the complex regulatory network through which miRNAs may regulate adverse cardiac remodeling. In fact, several studies have proposed miRNAs as therapeutic targets to treat deleterious ischemic effects in the heart ( 43 , 44 ), detrimental consequences of atherosclerosis ( 45 ), and to enhance cardio-protection after a myocardial I/R injury ( 46 ). Understanding the regulatory role of miRNAs could provide deeper insights into cells communication and the pathogenesis underlying adverse cardiac remodeling. Conclusions Our work highlights the pivotal role of immune cell-derived factors in cardiac remodeling after I/R. We demonstrated that myocardial injury induces distinct gene and miRNAs expression profiles within rat monocyte populations, reflecting their activated states, and further revealed that the secretome rich on miRNAs from these activated immune cells could directly modulate key fibrotic and apoptotic gene expression in cardiac fibroblasts and cardiomyocytes. Importantly, we identified specific miRNAs, namely miR-16-5p, miR-27a-3p, and miR-30b-5p, as regulators of these processes. These data were corroborated in humans, which showed a dysregulation of inflammation, fibrosis, and apoptosis-related genes in PBMCs from STEMI patients after the revascularization. Altogether, our findings underscore the translational relevance of immune cell-mediated gene, miRNAs regulation and communication with myocardial cells in adverse cardiac remodeling, suggesting novel targets for therapeutic interventions to mitigate cardiac remodeling and preventing progression to heart failure. Abbreviations I/R ischemia/reperfusion AMI acute myocardial infarction STEMI ST-segment elevation myocardial infarction COL1 collagen type 1 COL3 collagen type 3 TGFB1 transforming growth factor beta 1 APAF1 apoptotic protease activating factor-1 BCL2 B-cell lymphoma 2 CYCS cytochrome c somatic AKT2 AKT Serine/Threonine Kinase 2 HMOX1 Heme Oxygenase 1 MYC MYC Proto-Oncogene, BHLH Transcription Factor NFKB1 Nuclear Factor Kappa B Subunit 1 Declarations Ethics approval and consent to participate This study was conducted following the principles published by the declaration of Helsinki and its modification or similar ethical standards. The study was authorized by the local Ethics Committee on Human Research at the University Hospital “Virgen del Rocio” of Seville (Approvals no. 2013PI/096, 2018/352, 2021/03 and FIS-ISG-2024-01). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Supplementary information The online version contains supplementary material available at Funding Debora Falcon is supported by EMERGIA talent fellowship. This study was funded by “European Regional Development Fund; A way of making Europe,” and by the “European Union” and the Andalusia Government (grants numbers: ProyExcel_00530, PI-0034-2021, PI-0020-2024), the Institute of Carlos III (grant no. PI18/01197, PI23/01925), and the Spanish Ministry of Economy and Competitiveness (PID2022-136279NB-C22). Author Contribution E.B. and D.F. substantially and equally contributed to the acquisition, analysis and interpretation of all data in the manuscript. I.G.O. contributed to the acquisition and analysis of data. F.M.C. contributed to the analysis of gene expression data. G.B.E. contributed with the acquisition of human samples and analysis of experimental human data and provided fundings. I.V. contributed with advice assistance and funding. A.O.F. T.S. and R.D.T. contributed to the conception and design of the work, interpretation of data and provided fundings. T.S. and R.D.T wrote the original manuscript. All authors read and approved the submitted manuscript. Acknowledgement We thanks to A. Guisado and F. Guerrero-Marquez for providing peripheral blood human samples. We acknowledge the support of R. March from the Genomic’s facility, María José Castro from flow cytometry’s facility and Rocio Duran from the Histology’s facility at IBiS. Data Availability The datasets supporting the conclusions of this article are included within the article and its supplementary files. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9448122","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":634910653,"identity":"26f4393d-e8d1-4040-9672-0f3ebb4550ab","order_by":0,"name":"Elisa Bevilacqua","email":"","orcid":"","institution":"University of Seville","correspondingAuthor":false,"prefix":"","firstName":"Elisa","middleName":"","lastName":"Bevilacqua","suffix":""},{"id":634910654,"identity":"ee16c36b-0f4b-42ba-971d-fad21da97aed","order_by":1,"name":"Débora Falcón","email":"","orcid":"","institution":"University of Seville","correspondingAuthor":false,"prefix":"","firstName":"Débora","middleName":"","lastName":"Falcón","suffix":""},{"id":634910662,"identity":"66504ffb-6a0e-4ba4-b70c-6d5c49c0b3c1","order_by":2,"name":"Isabel Galeano-Otero1","email":"","orcid":"","institution":"University of Seville","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"","lastName":"Galeano-Otero1","suffix":""},{"id":634910664,"identity":"c1e19a62-45d5-4da9-9540-ff8955b053ec","order_by":3,"name":"Francisco Morón-Civanto","email":"","orcid":"","institution":"University of Seville","correspondingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"","lastName":"Morón-Civanto","suffix":""},{"id":634910667,"identity":"89943076-6f9b-450f-8bc9-4fdc51498afc","order_by":4,"name":"Gonzalo Barón-Esquivías","email":"","orcid":"","institution":"University of Seville","correspondingAuthor":false,"prefix":"","firstName":"Gonzalo","middleName":"","lastName":"Barón-Esquivías","suffix":""},{"id":634910669,"identity":"86dac7c7-41d3-4baf-a0b8-716d026618e1","order_by":5,"name":"Israel Valverde","email":"","orcid":"","institution":"University of Seville","correspondingAuthor":false,"prefix":"","firstName":"Israel","middleName":"","lastName":"Valverde","suffix":""},{"id":634910670,"identity":"8629266a-7823-4335-b11b-0f9848e793be","order_by":6,"name":"Antonio Ordóñez-Fernández","email":"","orcid":"","institution":"University of Seville","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Ordóñez-Fernández","suffix":""},{"id":634910671,"identity":"688f4402-e59a-46ca-a1bb-d09e24330214","order_by":7,"name":"Tarik Smani","email":"","orcid":"","institution":"University of Seville","correspondingAuthor":false,"prefix":"","firstName":"Tarik","middleName":"","lastName":"Smani","suffix":""},{"id":634910673,"identity":"b3d5835e-4e07-4961-bbf6-0739d1340d59","order_by":8,"name":"Raquel del Toro","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIie3PIQ7CMBTG8bcsoaYDW0HGFZogEAiu0mUCswSBqUBUjUsQjjHLW14CplyBDAMWHHMsI8FACjhEf6am//QrgOf9JRVge4YcEPR3CSA+E/tTAhwgyL8IRssU6aIPMbCoLOs1TYAtK2fStyeFaOdDCLuKooISw610JkJkEstcJRvikoKCFIjMPUyI2aVNTMhlWa+aYYOze1jzCjwTjAwFRsCHYfwk0Vr1+AvfTpOcZx8Slh4rrVUMvT1db4vxpMd27mGvOj/e9zzP8964A5mBSJXKA56SAAAAAElFTkSuQmCC","orcid":"","institution":"University of Seville","correspondingAuthor":true,"prefix":"","firstName":"Raquel","middleName":"del","lastName":"Toro","suffix":""}],"badges":[],"createdAt":"2026-04-17 10:54:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9448122/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9448122/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108976233,"identity":"13bee7b4-d452-491f-a9fa-2967be3407a0","added_by":"auto","created_at":"2026-05-11 11:00:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":281253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInflammatory cell subsets in human and rat peripheral blood and myocardium.\u003c/strong\u003e Bar graphs show levels of neutrophils (\u003cstrong\u003eA\u003c/strong\u003e), classical (\u003cstrong\u003eB\u003c/strong\u003e), intermediate (\u003cstrong\u003eC\u003c/strong\u003e) and non-classical monocytes (\u003cstrong\u003eD\u003c/strong\u003e) in healthy controls (blue, n = 33) and in STEMI patients (pink, n = 88) measured in peripheral blood at 0 h (before revascularization) and 6h, 1 month and 6 months (after revascularization). Bar graphs show levels of neutrophils (\u003cstrong\u003eE\u003c/strong\u003e), CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e (\u003cstrong\u003eF\u003c/strong\u003e) and CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e monocytes (\u003cstrong\u003eG\u003c/strong\u003e) in peripheral blood of control (dark green, n=14), SHAM (light green, n=4-11) and I/R (orange, n=5-12) rats at 72h, 1 and 4 weeks. Immunofluorescence staining of M1 macrophages with CD68 (green) and nuclei with DAPI (blue) and quantification of CD68\u003csup\u003e+\u003c/sup\u003e cell percentage in the middle papillary region of hearts of SHAM (green, n=3-14) and I/R (orange, n=5-16) rats at 72h (\u003cstrong\u003eH\u003c/strong\u003e), 1 (\u003cstrong\u003eI\u003c/strong\u003e) and 4 weeks (\u003cstrong\u003eJ\u003c/strong\u003e) after the intervention. Data are represented as mean ± S.D. (*), (**), (***) and (****) indicate significance at p \u0026lt; 0.05 and p\u0026lt;0.01, p\u0026lt;0.001, p\u0026lt;0.0001, respectively, and # (p\u0026lt;0.001) indicates significant differences between control and STEMI. STEMI: ST-elevation myocardial infarction; I/R: ischemia and reperfusion. Group comparisons were performed using Kruskal-Wallis test with multiple comparisons for A- E graphs, and ordinary one-way ANOVA with multiple comparisons for F and G graphs. Differences between two groups were assessed using the unpaired t-test for H and I panels, and Mann-Whitney test for J panel.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9448122/v1/4c029a5c5e9dcdfb1d335d9b.png"},{"id":108975932,"identity":"09d6734c-fdd2-4001-8c04-e97a6d6ee4e7","added_by":"auto","created_at":"2026-05-11 10:58:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":341346,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenes differentially expressed between SHAM and I/R rats in sorted monocytes and gene enrichment analysis. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePrincipal component analysis (PCA) of genes in SHAM (purple and green, n=4) and I/R (red and blue, n=4) of CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e (left side) and CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e (right side) 24h after I/R procedure. Number of differently expressed genes (\u003cstrong\u003eB\u003c/strong\u003e) in the two subtypes of monocytes. Volcano plots of –1.5 \u0026gt; fold change \u0026gt; 1.5 value of genes in CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e (\u003cstrong\u003eC\u003c/strong\u003e) and CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e monocytes (\u003cstrong\u003eD\u003c/strong\u003e) from I/R (n=4) \u003cem\u003evs\u003c/em\u003e. SHAM (n=4) 24h after the intervention. Panther gene analysis of the main pathways in the two types of monocytes (\u003cstrong\u003eE-F\u003c/strong\u003e). Bubble plot of pathways different enriched by GSEA analysis in CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e (\u003cstrong\u003eG\u003c/strong\u003e) and CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e (\u003cstrong\u003eH\u003c/strong\u003e) monocytes. I/R: ischemia and reperfusion.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9448122/v1/90f73d01aefeb0587de39a6d.png"},{"id":108976093,"identity":"a799c0ad-140c-40c7-866e-ee079d07e7b4","added_by":"auto","created_at":"2026-05-11 10:59:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":374600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibrotic markers in cardiac populations stimulated with neutrophils and monocytes from I/R rats.\u003c/strong\u003e Experimental protocol for cardiac cell populations stimulated with immune cells (\u003cstrong\u003eA\u003c/strong\u003e). Fibrosis in rat infarcted hearts with\u003cstrong\u003e \u003c/strong\u003eMasson’s trichrome staining in the apex and middle papillary region of SHAM (\u003cstrong\u003eB\u003c/strong\u003e) and I/R (\u003cstrong\u003eC\u003c/strong\u003e) rats. Healthy tissue is stained in red, while fibrotic tissue in the infarcted zone is stained in blue. Bar graphs show fibrotic genes \u003cem\u003eCol1\u003c/em\u003e, \u003cem\u003eCol3\u003c/em\u003e and \u003cem\u003eTgfb1\u003c/em\u003e (\u003cstrong\u003eD\u003c/strong\u003e) and apoptotic genes \u003cem\u003eApaf1\u003c/em\u003e, \u003cem\u003eBcl2\u003c/em\u003e and \u003cem\u003eCycs\u003c/em\u003e (\u003cstrong\u003eE\u003c/strong\u003e) expression levels measured by RT-qPCR normalized to SHAM (represented with the grid line, n=6-7) of cardiomyocytes and fibroblasts stimulated with monocytes and neutrophils supernatant from I/R rats (in orange, n=5-7). Gene's relative expression was calculated using the 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e method after normalization to the internal control 18S. Data are relative expression of mean of fold change ± S.D. Group comparisons were performed using Kruskal-Wallis with multiple comparisons for D and E panels. I/R: ischemia and reperfusion; \u003cem\u003eCol1\u003c/em\u003e: collagen type 1; \u003cem\u003eCol3\u003c/em\u003e: collagen type 3; \u003cem\u003eTgfb1\u003c/em\u003e: transforming growth factor beta 1; \u003cem\u003eApaf1\u003c/em\u003e: apoptotic protease activating factor-1; \u003cem\u003eBcl2\u003c/em\u003e: B-cell lymphoma 2; \u003cem\u003eCycs\u003c/em\u003e: cytochrome c somatic. # indicates significance between I/R and SHAM at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9448122/v1/25e8770b1a089a2466e60592.png"},{"id":108976098,"identity":"0f7b61f1-43ba-4edf-95a3-b1cb528cc05a","added_by":"auto","created_at":"2026-05-11 10:59:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":335538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emicroRNAs differentially expressed between SHAM and I/R rats in sorted monocytes and their regulated genes. \u003c/strong\u003ePrincipal component analysis (PCA) of microRNAs (\u003cstrong\u003eA\u003c/strong\u003e) in SHAM (purple and green, n=4) and I/R (red and blue, n=4) of CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e (left side) and CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e (right side) 24h after the intervention. (\u003cstrong\u003eB\u003c/strong\u003e) Number of differently expressed genes in the two subtypes of monocytes. Volcano plots of –1.5 \u0026gt; fold change \u0026gt; 1.5 value of genes in CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e (\u003cstrong\u003eC\u003c/strong\u003e) and CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e monocytes (\u003cstrong\u003eD\u003c/strong\u003e) from I/R (n=4) \u003cem\u003evs.\u003c/em\u003e SHAM (n=4) 24h after the intervention. (\u003cstrong\u003eE\u003c/strong\u003e) List of microRNAs from \u003cem\u003ein silico\u003c/em\u003e study in CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e monocytes, regulating apoptotic and fibrotic genes studied in the cardiac populations. I/R: ischemia and reperfusion. \u003cem\u003eCol1a\u003c/em\u003e: collagen type 1a; \u003cem\u003eCol3a\u003c/em\u003e: collagen type 3a; \u003cem\u003eTgfb1\u003c/em\u003e: transforming growth factor beta 1; \u003cem\u003eApaf1\u003c/em\u003e: apoptotic protease activating factor-1; \u003cem\u003eBcl2\u003c/em\u003e: B-cell lymphoma 2; \u003cem\u003eCycs\u003c/em\u003e: cytochrome c somatic;\u003cem\u003e IL1b\u003c/em\u003e: interleukin 1 beta;\u003cem\u003e IL6\u003c/em\u003e:\u003cem\u003e \u003c/em\u003einterleukin 6;\u003cem\u003e IL17a\u003c/em\u003e: interleukin 17a; miR: microRNA.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9448122/v1/6577abc92859cc68568a0510.png"},{"id":108976234,"identity":"e0367854-e87a-42ac-bdd4-bc694141738d","added_by":"auto","created_at":"2026-05-11 11:00:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":182718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emicroRNAs expression in CD43\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ehigh\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e His48\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003elow\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e monocytes in rats. \u003c/strong\u003eRT-qPCR microRNA validation (miR-16-5p, \u003cstrong\u003eA\u003c/strong\u003e; miR-27a-3p, \u003cstrong\u003eB\u003c/strong\u003e; miR-29b-3p, \u003cstrong\u003eC\u003c/strong\u003e; miR-30b-5p, \u003cstrong\u003eD\u003c/strong\u003e; miR-30e-5p, \u003cstrong\u003eE\u003c/strong\u003e; miR-194-5p, \u003cstrong\u003eF; \u003c/strong\u003emiR-18a-5p, \u003cstrong\u003eG;\u003c/strong\u003e miR-125a-5p, \u003cstrong\u003eH; \u003c/strong\u003emiR-222-3p, \u003cstrong\u003eI\u003c/strong\u003e) in CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e monocytes from SHAM (purple dots, n=4-5) and I/R (blue dots, n= 4-5) rats 24h after the intervention. miRNA's relative expression was calculated using the 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e method after normalization to the internal control U6. Data are relative expression of mean of fold change ± S.D. (*), (**) and (***) indicate significance at p \u0026lt; 0.05, p \u0026lt; 0.01 and p\u0026lt;0.001, respectively. Differences between two groups were assessed using the unpaired t-test for A- E graphs and G and I, and Mann-Whitney test for F and H graphs. I/R: ischemia and reperfusion; miR: microRNA.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9448122/v1/f14fbb1028e3970dcece99f0.png"},{"id":108976173,"identity":"372074e1-a236-4f9b-b7d7-bdf1cdf5c829","added_by":"auto","created_at":"2026-05-11 10:59:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":323537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMimic regulation of fibrotic and apoptotic genes in cardiomyocytes and fibroblasts of rats. \u003c/strong\u003eQuantification of mRNA expression of fibrotic genes \u003cem\u003eCol1\u003c/em\u003e, \u003cem\u003eCol3\u003c/em\u003e and \u003cem\u003eTgfb1\u003c/em\u003e (\u003cstrong\u003eA\u003c/strong\u003e) and apoptotic genes \u003cem\u003eApaf1\u003c/em\u003e, \u003cem\u003eBcl2\u003c/em\u003e and \u003cem\u003eCycs\u003c/em\u003e (\u003cstrong\u003eB\u003c/strong\u003e) using RT-qPCR in NRVMs (n=6) and fibroblasts (n=5) when transfected with scramble (light beige for controls and dark beige for I/R), miR-16-5p mimic (violet for I/R) and miR-30b-5p mimic (pink for I/R). Gene's relative expression was calculated using the 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e method after normalization to the internal control 18S. Data are relative expression of mean of fold change ± S.D. (*), (**) and (****) indicate significance at p \u0026lt; 0.05, p \u0026lt; 0.01 and p\u0026lt;0.0001, respectively. Group comparisons were performed using ordinary one-way ANOVA with multiple comparisons for A and B panels, and Kruskal-Wallis test with multiple comparisons for Cycs in cardiomyocytes in B panel. I/R, ischemia and reperfusion; NRVCs, neonatal rat ventricular cardiomyocytes; miR: microRNA; \u003cem\u003eCol1:\u003c/em\u003e collagen type 1; \u003cem\u003eCol3\u003c/em\u003e: collagen type 3; \u003cem\u003eTgfb1\u003c/em\u003e: transforming growth factor beta 1; \u003cem\u003eApaf1\u003c/em\u003e: apoptotic protease activating factor-1; \u003cem\u003eBcl2\u003c/em\u003e: B-cell lymphoma 2; \u003cem\u003eCycs\u003c/em\u003e: cytochrome c somatic; miR: microRNA.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9448122/v1/4b9ef49385a1d9c686dd945b.png"},{"id":108978804,"identity":"5a8be14b-3253-4292-9922-7693dff68a22","added_by":"auto","created_at":"2026-05-11 11:48:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2290632,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9448122/v1/35cdc405-ffbb-449c-84c6-775583b55ced.pdf"},{"id":108976174,"identity":"d6e67591-fb18-4265-818a-928b1b2dada0","added_by":"auto","created_at":"2026-05-11 10:59:21","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":111934,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-9448122/v1/8c176692c9c0102ff0a5e9b2.png"},{"id":108976097,"identity":"aa31c957-4363-4b16-b4c3-698d53397245","added_by":"auto","created_at":"2026-05-11 10:59:10","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2361125,"visible":true,"origin":"","legend":"","description":"","filename":"Supplemental.docx","url":"https://assets-eu.researchsquare.com/files/rs-9448122/v1/e6cae4eca924a7628ab38082.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of pro-inflammatory monocyte subsets and their microRNAs regulation after an ischemia/reperfusion myocardial injury","fulltext":[{"header":"Background","content":"\u003cp\u003eAn ischemic event resulting from coronary artery occlusion initiates a cascade of structural, functional, and metabolic disturbances that impair myocardial performance. Primary percutaneous coronary intervention (PPCI) remains the most effective treatment for limiting cardiac cell death and reducing the risk of adverse cardiac events following a severe ST-segment elevation myocardial infarction (STEMI) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). However, despite timely PPCI, a substantial proportion of STEMI patients still develop adverse cardiac remodeling post-PPCI (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), involving structural and molecular changes that compromise cardiac performance and contribute to the progression toward heart failure (HF) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the various pathophysiological processes triggered by STEMI, fibrosis and apoptosis are key contributors to adverse remodeling following the ischemic event (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Fibrosis results in excessive extracellular matrix deposition, impairing myocardial elasticity and function, while apoptosis contributes to the loss of cardiomyocytes, weakening the contractile capacity of the heart and exacerbating cardiac dysfunction. Peripheral blood-derived monocytes and neutrophils play a pivotal role in both cardiac injury and repair processes, facilitating wound healing through temporally distinct actions (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Neutrophils are rapidly recruited to the infarcted myocardium, initiating a pro-inflammatory response, while monocytes infiltrate later and differentiate into macrophages that support tissue repair and promote resolution of inflammation (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, excessive or prolonged activation of these innate immune cells can exacerbate tissue damage, contributing to adverse remodeling and increasing the risk of HF and other cardiac events (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Nevertheless, the exact mechanisms through which innate immunity impacts ventricular adverse remodeling and progression to HF remain incompletely understood.\u003c/p\u003e \u003cp\u003eMonocytes represent a heterogeneous cell population classified into three subsets in humans, each characterized by specific phenotypic markers and functional roles during inflammation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In rats, monocytes immunodefined as CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e and CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e are thought to correspond to the classical CD14\u003csup\u003e++\u003c/sup\u003eCD16\u003csup\u003e\u0026minus;\u003c/sup\u003e and non-classical CD14\u003csup\u003e+\u003c/sup\u003e CD16\u003csup\u003e++\u003c/sup\u003e human monocytes subsets, respectively (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Despite these proposed analogies, the precise functional roles of these rat monocyte subsets remain to be elucidated (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), highlighting the need for further phenotypic and functional characterization, especially in the context of inflammatory and ischemic conditions. Our previous studies demonstrated that inflammatory cell populations can produce a repertoire of pro-inflammatory mediators as chemokines, cytokines, microRNAs (miRNAs), as well as factors involved in the resolution of inflammation that could be used as prognostic markers (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). miRNAs belong to non-coding RNA family extensively involved in cardiovascular diseases prognosis and regulation (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Growing evidence further supports the role of miRNAs not only as biomarkers of cardiovascular disease but also as key post-transcriptional regulators of gene expression implicated in cardiac remodeling (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we investigated the contribution of ischemia/reperfusion (I/R) rats monocytes and neutrophils to adverse cardiac remodeling. Specifically, we examined their capacity to modulate the expression of fibrosis- and apoptosis-related genes in cardiomyocytes and fibroblasts. Furthermore, we analyzed the dysregulation of miRNAs in sorted rat monocytes and explored their potential regulatory roles in gene networks associated with fibrosis and apoptosis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis study was conducted following the principles published by the declaration of Helsinki and its modification or similar ethical standards. The study was authorized by the local Ethics Committee on Human Research at the University Hospital \u0026ldquo;Virgen del Rocio\u0026rdquo; of Seville (Approvals no. 2013PI/096, 2018/352, 2021/03 and FIS-ISG-2024-01).\u003c/p\u003e\n\u003cp\u003eExperiments with animals were performed in accordance with the recommendations of the Royal Decree 53/2013 in agreement to the Directive 2010/63/EU of the European Parliament and approved by the Animal Research Committee of the University of Seville (18/08/2020/097 and 22/07/2024/110).\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eStudy with patients\u003c/h2\u003e\n\u003cp\u003eAll the subjects of this study were voluntary participants and signed an informed consent form. A cohort of 121 patients were prospectively recruited between March 11th 2016 and September 1st 2023. The participants were divided into two groups: 33 healthy volunteers (49\u0026thinsp;\u0026plusmn;\u0026thinsp;12 years; 48.5% male sex) were persons who do not suffer arterial hypertension, dyslipidemia or diabetes mellitus type II, do not smoke and without diagnosed coronary disease, and 88 who were diagnosed with STEMI (58\u0026thinsp;\u0026plusmn;\u0026thinsp;10 years; 84.1% male sex) and treated with a primary percutaneous coronary intervention (PPCI) through the right radial artery at the University Hospital \u0026ldquo;Virgen del Rocio\u0026rdquo; of Seville. Clinical and demographical information were collected at the admission in the hospital and during each follow-up visit, scheduled at 1 and 6 months after the hospital discharge. 51% completed the follow-up. The inclusion criteria were, age less than 75 years; patients with STEMI due to occlusion of the left descending artery with an epicardial blood flow TIMI (Thrombolysis in Myocardial Infarction) grade 0\u0026ndash;1 in the initial angiogram treated with PPCI, with the onset of symptoms less than 12h before the angioplasty. The exclusion criteria were, ischemic heart disease history, a\u0026thinsp;\u0026lt;\u0026thinsp;30 ml/min glomerular filtration rate, and a TIMI flow grade over 1.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eRat cardiac surgery animal model\u003c/h3\u003e\n\u003cp\u003eMale healthy Wistar rats weighing 250\u0026thinsp;\u0026plusmn;\u0026thinsp;50 g were anesthetized with intraperitoneal mixture of 50 mg/kg ketamine plus 8 mg/kg xylazine and maintained in 2% sevoflurane and oxygen described previously (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e). A left thoracotomy was performed between the third and fourth rib, followed by a pericardiotomy. The left coronary artery was occluded with a 5\u0026thinsp;\u0026minus;\u0026thinsp;0 ProleneTM silk suture (Ethicon, Spain) and the knot was performed over a tube placed into the suture releasing the occlusion after 40 min to perform an ischemia/reperfusion model. The left coronary artery occlusion was confirmed by visual observation of cyanosis and ST-segment elevation by continuous ECG monitoring. The chest cavity was closed, and the air was expelled from the chest. Analgesia was induced with meloxicam (1 mg/kg) administered subcutaneously. Rats were left on a heating pad until fully conscious recovery; mortality during the interventions was 3%. For this study, we considered the following experimental groups: I/R, ischemia and reperfusion model with left coronary artery ligation during 40 min, and SHAM, where left thoracotomy was performed without touching the heart to remove the basal inflammation due to the intervention, and control rats. Blood samples were obtained at different time points (24h, 72h, 1 week, 4 weeks), while tissues (heart and spleen) were extracted at the time point of 72h, 1 and 4 weeks.\u003c/p\u003e\n\u003ch3\u003eBlood and tissue samples: extraction and preparation\u003c/h3\u003e\n\u003cp\u003eHuman peripheral blood samples were collected into ethylenediaminetetraacetic acid (EDTA)-coated tubes (BD Vacutainer\u0026reg; K2E; BD Biosciences, USA) and extracted before revascularization (STEMI 0h) and after culprit vessel opening (STEMI 6h, 1 and 6 months). 100 \u0026micro;l of blood was incubated during 30 min at room temperature (RT) with CD11b, CD14 and CD16 and CD66b antibodies (APC Mouse Anti-Human CD11b/Mac-1 clone ICRF; FITC Mouse Anti-Human CD14 clone M5E2; PE Mouse Anti-Human CD16 clone 3G8; PerCP-Cy\u0026trade;5.5 Mouse Anti-Human CD66b clone G10F5, respectively; BD Pharmingen, USA). Then, red blood cells were lysed with BD FACS\u0026trade; Lysing Solution 10X Concentrate (BD Biosciences, USA) following manufacture instructions and washed with phosphate buffered saline (PBS 1X; GIBCO, USA) and centrifuged. The pellet was resuspended in 300 \u0026micro;l of PBS 1X and analyzed by flow cytometry.\u003c/p\u003e\n\u003cp\u003eHuman peripheral blood mononuclear cells (PBMCs) were extracted as previously described (\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e). Briefly, 3 ml of EDTA peripheral blood was diluted with PBS 1x 1:1 and deposited above Ficoll (Lymphosep- Lymphocyte Separation Medium, MP Biomedical, USA) following manufacture instructions. After centrifugation at RT for 20 min at 1600 rpm without brake, PBMCs were recollected from the above phase and washed with PBS 1X. Supernatant was removed, and the pellet was frozen and keep to -20\u0026ordm;C for RNA extraction.\u003c/p\u003e\n\u003cp\u003eRats red blood cells were lysed with distilled water during 1 min 30 sec and the lysis was stopped with 1.8% NaCl solution. Spleen homogenate was obtained by half of the organ shredding and red blood cells were lysed with distilled water during 1 min. Cells were incubated with 1 \u0026micro;l of His48, CD11b and CD43 antibodies (Mouse Anti- Rat Granulocyte Marker His48 FITC, Invitrogen, USA; Mouse Anti- Rat CD11b APC, BD Pharmingen, USA; Mouse Anti- Rat CD43 Alexa Fluor 594, R\u0026amp;D systems, USA).\u003c/p\u003e\n\u003cp\u003eRat hearts were directly included in Tissue-Tek\u0026reg; O.C.T. Compound (Sakura Finetek, USA) and frozen for immunofluorescence or fixed with formalin and embedded in paraffine following the dehydration process for trichrome Masson\u0026rsquo; staining.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry measurements\u003c/h3\u003e\n\u003cp\u003eHuman blood samples were acquired in a Canto II flow cytometer (BD Biosciences, USA), while the rat samples were analyzed at LRS II Fortessa flow cytometer (BD Biosciences, USA) and sorted at MoFlo Astrios FACS (Beckman Coulter, USA). Sample acquisition and analysis was performed with the FACS-Diva software 8.0 (BD Biosciences, USA). The inflammatory human cell populations analyzed were, neutrophils (CD16\u003csup\u003e++\u003c/sup\u003eCD66b\u003csup\u003e+\u003c/sup\u003e), eosinophils (CD16\u003csup\u003e+\u003c/sup\u003eCD66b\u003csup\u003e\u0026minus;\u003c/sup\u003e) and monocyte subsets. Monocyte subgroups were classified as CD14\u003csup\u003e++\u003c/sup\u003e/CD16\u0026ndash; (classical), CD14\u003csup\u003e++\u003c/sup\u003e/CD16\u003csup\u003e+\u003c/sup\u003e (intermediate), and CD14\u003csup\u003e+\u003c/sup\u003e/CD16\u003csup\u003e++\u003c/sup\u003e (non-classical). The inflammatory rat cell populations analyzed were, neutrophils (FSC/SSC, CD11b\u003csup\u003e+\u003c/sup\u003e), and the two monocyte subsets: CD11b\u003csup\u003e+\u003c/sup\u003e CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e (non-classical) and CD11b\u003csup\u003e+\u003c/sup\u003e CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e (classical) monocytes.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence and histology staining\u003c/h3\u003e\n\u003cp\u003eFor trichrome Masson staining, hearts were fixed in formalin, dehydrated, embedded in paraffin and sectioned at 6 \u0026micro;m using a microtome. Tissue sections were then stained according to manufacturer's instructions to visualize fibrosis. Images were acquired using an Olympus BX-61 direct microscope and analyzed with a proper pipeline to detect fibrosis at CellProfiler software.\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence experiments, hearts were sliced at the cryostat with a width section of 6 \u0026micro;m and then were fixed with formalin for 30 min. Slices were washed with PBS 1X and permeabilized with PBS 1X- 0.3% Triton (Triton X100, Sigma) during 15 min. Subsequently, to block unspecific binding of the antibodies, sections were incubated with PBS 1X- 0.1% Triton- 10% Goat serum (Gibco, USA)- 1% BSA (Bovine Serum Albumin, A2153-100G, Sigma, USA) for 1h. Tissues were incubated with appropriate diluted antibodies (CD163, ED2 clone 1:100; CD68, ED1 clone 1:100, Biorad, USA) in blocking solution in a humidified chamber overnight at 4\u0026deg;C. Slices were washed and then incubated with secondary antibodies (Alexa Fluor 488 goat anti-mouse, Alexa Fluor 594 goat anti-mouse, 1:400, Thermo Fisher Scientific, USA) in blocking solution for 3h at RT in the dark. Finally, nuclei were stained with DAPI (4',6-diamidin-2-fenilindolo, Sigma, USA) and coverslip were mounted with a drop of mounting medium DAKO Fluorescence Mounting Medium (Dako, USA).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eIsolation of adult cardiac cells\u003c/h2\u003e\n\u003cp\u003eCardiac populations, enriched in fibroblasts and cardiomyocytes, were isolated using a standard enzymatic digestion (Collagenase type II, 251 IU/mL; Worthington Biochemical, USA) as described previously (\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e). Briefly, hearts were removed and perfused on a Langendorff perfusion device. After perfusion, hearts were left in enzyme solution supplemented with 2 g/L BSA. Isolated cells were then filtered, centrifuged and suspended in Tyrode solution containing (in mM): 130 NaCl, 1 CaCl\u003csub\u003e2\u003c/sub\u003e, 0.5 MgCl\u003csub\u003e2\u003c/sub\u003e, 5.4 KCl, 22 glucose, 25 HEPES, 0.4 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 5 NaHCO\u003csub\u003e3\u003c/sub\u003e; pH was adjusted to 7.4 with NaOH. Cardiac populations were obtained with differential centrifugation (200 rpm for 1 min for cardiomyocytes, 1500 rpm for 5 min for fibroblasts). Cells were cultured on laminin (L2020, Sigma, USA) pretreated plates in control solution containing RPMI 1640 medium (Corning, USA) supplemented with 8% fetal bovine serum (FBS; Gibco, USA), 10% Streptomycin-Penicillin (10.000 units, Capricorn Scientific, USA) and 10% L- glutamine (200mM, Sigma, USA) at 37\u0026ordm;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e\u0026nbsp;\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cstrong\u003eGranulocytes secretome medium preparation and cardiac cells stimulation\u003c/strong\u003e\u003c/div\u003e\n\u003cp\u003eGranulocytes obtained from peripheral blood of SHAM and I/R rats, 72h after the intervention, were used to stimulate myocardial populations. Neutrophils and monocytes were isolated with 10% dextran (Sigma, USA) in a proportion of 2:15. After 30 min of decantation, supernatant was collected and deposited above Ficoll (Lymphosep- Lymphocyte Separation Medium, MP Biomedical, USA). Monocytes were obtained from the above Ficoll phase, while neutrophils were collected from the pellet. The two populations were cultured with RPMI 1640 medium (Corning, USA) supplemented with B27 (Corning, USA), 10% Streptomycin-Penicillin (10.000 units, Capricorn Scientific, USA) and 10% L- glutamine (200mM, Sigma, USA) for 24h. Then, supernatant was collected and added in proportion 1:1 to fibroblasts and cardiomyocytes obtained before. Cells were cultured with the mixed medium containing 4% of FBS. 24h after the treatment, total RNA from fibroblasts and cardiomyocytes was collected.\u003c/p\u003e\n\u003ch3\u003eNeonatal cardiac cell culture and miRNA transfection\u003c/h3\u003e\n\u003cp\u003eFor cell transfection analysis we used neonatal rat ventricle myocytes (NRVMs) and cardiac fibroblasts. They were isolated from hearts of 1\u0026ndash;3 days old Wistar rats using trypsin as described previously (\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e). Following trypsinization, fibroblasts were separated from NRVMs based on their differential adhesion properties. Cardiac fibroblasts adhered to the culture dishes within 1h, while NRVMs exhibited poor attachment to the coated surface during this short period. After the 1h incubation, the supernatant containing the unattached cardiomyocytes was collected, and the fibroblasts remained attached to the plate. NRVMs were cultured in DMEM/M199 (4:1) (Gibco, USA) supplemented with 10% horse serum, 5% FBS, 1% glutamine, 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin, while cardiac fibroblasts were grown in DMEM supplemented with 10% FBS, 1% glutamine, 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin.\u003c/p\u003e\n\u003cp\u003eNRVMs and cardiac fibroblasts were transfected with miRNA mimics at 70% confluence, 48h post-isolation, following the manufacturer's instructions using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, USA). The cells were incubated for an additional 48h before subsequent experiments. Briefly, 6 \u0026micro;l of Lipofectamine was diluted in 250 \u0026micro;l of Opti-MEM\u0026reg; Medium (Gibco, USA), then 4,5 \u0026micro;l of miR-16 mimic, miR-27a mimic or miR-30b mimic (10 \u0026micro;M) (Thermo Fisher Scientific, USA) was diluted in 250 \u0026micro;l of Opti-MEM\u0026reg; Medium. The solutions were mixed in a 1:1 ratio and incubated for 10 min at RT. Then, mix was added to cells.\u003c/p\u003e\n\u003cp\u003eIschemia/reperfusion (I/R) was performed 24h post-transfection using a simulated ischemic solution (in mM): 142 NaCl, 3.6 KCl, 1.2 MgCl\u003csub\u003e2\u003c/sub\u003e, 1.8 CaCl\u003csub\u003e2\u003c/sub\u003e, 5 NaHCO\u003csub\u003e3\u003c/sub\u003e, 20 Hepes, 20 Lactate-Na, 20 sucrose; pH 6.22, in a hypoxia chamber (1% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e), for 3h. Reperfusion/reoxygenation started when the ischemic solution was removed and cells were placed in an incubator at 21% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e in DMEM/M199 (4:1) supplemented with 0.4% horse serum, 0.07% FBS, 100 U/ml penicillin and 100 \u0026micro;g/ml streptomycin.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eMiRNAs and genes Array\u003c/h2\u003e\n\u003cp\u003eArrays were performed from sorted monocytes of SHAM and I/R rats 24h after the intervention. Total RNA was extracted with miRNAeasy mini kit (Qiagen, Germany) according to the manufacturer\u0026rsquo;s instructions. The total cDNA was labeled using the FlashTag\u0026reg; Biotin HSR labeling Kit (Thermo Fisher Scientific, USA). GeneChip\u0026reg; miRNA 4.0 arrays (Thermo Fisher Scientific, USA) were used to analyze miRNAs, while Clariom S Assay Rat (Thermo Fisher Scientific, USA) was used to study gene expression. Washing, staining (GeneChip\u0026reg; Fluidics Station 450, Thermo Fisher Scientific, USA), and scanning (GeneChip\u0026reg; Scanner 3000, Thermo Fisher Scientific, USA) were done following manufacturer\u0026rsquo;s protocol. Briefly, importing CEL file, the analysis of miRNA level RMA+DABG-All and exporting the results were done using Transcriptome Analysis Console (TAC) 4.0 software (Thermo Fisher Scientific, USA). A comparative analysis between SHAM rats and I/R rats was carried out using fold-change of over \u0026plusmn;\u0026thinsp;1.5 with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e analysis of miRNAs regulating apoptosis and fibrosis related genes was performed with TAC 4.0 software. The different expressed miRNAs were studied with miRDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.miRDB.com\u003c/span\u003e\u003c/span\u003e) and TargetScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.targetscan.org\u003c/span\u003e\u003c/span\u003e) to evaluate possible target genes. Later, the target genes were filtered with GeneVenn (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.genevenn.sourceforge.net\u003c/span\u003e\u003c/span\u003e). Target genes implicated in different biological pathways were studied with Panther Gene (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.pantherdb.org/\u003c/span\u003e\u003c/span\u003e). Enrichment analysis for genes was performed firstly with Gene Ontology (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.geneontology.org\u003c/span\u003e\u003c/span\u003e) and then with GSEA 4.3.2 (Gene Set Enrichment Analysis), using Biological Processes C5 library 6.2 version.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eRNA extraction and RT-qPCR\u003c/h2\u003e\n\u003cp\u003eFor miRNA analysis, total RNA was extracted with miRNAeasy mini kit (Qiagen, Germany) according to the manufacturer\u0026rsquo;s instructions. To perform RT-qPCR, RNA was retrotranscribed to cDNA with miRCURY LNA RT Kit (Qiagen, Germany) for miRNAs detection. PCR mix for miRNAs included miRCURY LNA SYBR Green PCR kit (Qiagen, Germany) and hsa-miR-16-5p, hsa-miR-18a-5p, hsa-miR-27a-3p, hsa-miR-29b-3p, hsa-miR-30b-5p, hsa-miR-30e-5p, hsa-miR-125a-5p, hsa-miR-194-5p, hsa-miR-222-3p miRCURY LNA miRNA PCR Assay. Data analysis was made with QuantStudio\u0026trade; Real-Time PCR Software (Thermo Fisher Scientific, USA). Fold change quantification was calculated using the comparative cycle threshold (CT) method, using 18S and U6 as endogenous control.\u003c/p\u003e\n\u003cp\u003ePrime PCR was used as described previously (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e). Briefly, the retrotranscription was performed according to the manufacture instruction with iScript\u0026trade; Advanced cDNA Synthesis Kit (1725037, Biorad, USA). Apoptosis and survival Tier 1 H384 384 (10040258, Biorad, USA) was performed with SsoAdvanced Universal SYBR Green Supermix (1725270 EDU, Biorad, USA). The studied genes are listed in Supplemental Table\u0026nbsp;1.\u003c/p\u003e\n\u003cp\u003eFor gene analysis, RNA from patients\u0026rsquo; PBMCs were extracted with miRVana (AM1560, Thermo Fisher Scientific, USA), while for cardiac populations NucleoSpin RNA (740955.50.00, Macherey-Nagel, USA) was used. Retrotranscription from 1 \u0026micro;g of mRNAs was performed with iScript cDNA Synthesis Kit (1708891, Biorad, USA) for all the samples and cDNA was diluted 1:10. qPCR was performed in a FrameStar 384 Well PCR Plate (4titude, BIOK\u0026eacute;, Leiden, the Netherlands). PCR mix included SYBR Green reactive (iTaq\u0026trade; Universal SYBR Green Supermix; Biorad, USA) and specific oligos for each gene, listed in Supplemental Table\u0026nbsp;2. RT-qPCR were performed on the Applied Biosystems Viia7 Real-Time PCR System (Thermo Fisher Scientific, USA). The thermal cycling conditions were as follows: 95\u0026deg;C for 20 sec followed by 45 cycles of 95\u0026deg;C for 1 sec and 60\u0026deg;C for 20 sec. Expression data were calculated like fold change obtained with the comparative cycle threshold CT (\u0026Delta;\u0026Delta;CT) method, using 18S as endogenous control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n\u003cp\u003eStatistical analysis were performed with GraphPad (GraphPad Software, USA). Results are presented as mean and SD. Shapiro-Wilk was used as normality test. To compare normal data of 2 or more groups the t-student or the ordinary one-way ANOVA were used respectively, while for non-normal distributed data we used Mann-Whitney and Kurskall-Wallis test, respectively. Statistical significant differences were considered when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eInflammatory cells subsets dynamics in STEMI patients and in I/R rats\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn order to study the level of inflammatory cells in humans after an ischemic insult, we recruited 121 participants, healthy volunteers without diagnosed coronary disease and 88 who were diagnosed with a ST-segment elevation myocardial infarction (STEMI) and treated with a Primary percutaneous coronary intervention (PPCI). STEMI patients showed an accused neutrophilia, before (0h), 6h after the revascularization and 1 month later the ischemic event, during the follow-up, compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Levels of neutrophils decreased 6 months after PPCI, approaching the levels analyzed in the control group. STEMI patients also showed significantly increased levels of classical monocytes at 6h, (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), of intermediate monocytes at 0h and 6h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and non-classical monocytes 1 month after PPCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) compared to the control group.\u003c/p\u003e \u003cp\u003eTo complement the clinical findings, we evaluated inflammatory cell changes in a rat model of ischemia/reperfusion (I/R). We quantified neutrophils and monocytes levels in peripheral blood, from SHAM, I/R and rats without any intervention (Supplemental Fig.\u0026nbsp;1A-C), at different time points, 72h, 1 and 4 weeks after surgery procedures.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G, neutrophil levels augmented 72 h after the intervention in I/R rats, compared to SHAM operated rats, while 1 week after the operation this increase was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The analysis of monocytes sub-population indicates that I/R rats showed a marked increase in the level of CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e and CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e, especially 1 week post-surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). By 4 weeks, I/R rats showed a significant reduction in monocyte and neutrophils levels, suggesting resolution of the inflammatory phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the contribution of extramedullary hematopoiesis after the myocardial infarction, we analyzed immune cell populations in the spleen of I/R rats. We observed no significant change in neutrophil levels in I/R rats, except for a significant reduction at 72h. (Supplemental Fig.\u0026nbsp;1D). Regarding the two monocytes subsets, they remained unchanged in I/R rats across all time points (Supplemental Fig.\u0026nbsp;1E, 1F).\u003c/p\u003e \u003cp\u003eCollectively, these data confirm that the inflammatory response following an ischemic event in rats involves dynamic alterations in circulating and/or splenic neutrophils and monocytes, similar to STEMI patients.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCardiac macrophage infiltration after ischemic injury\u003c/h2\u003e \u003cp\u003ePeripheral blood monocytes infiltrate and differentiate into phagocytic macrophages in the heart after an ischemic event. Considering monocyte levels, we examined macrophage heart infiltration after I/R injury. Proinflammatory M1 macrophages, identified by CD68\u003csup\u003e+\u003c/sup\u003e expression, were absent in SHAM hearts but were detected in I/R rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-J). These cells were localized specially within the infarcted area at 72h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) and 1 week (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI) after the procedure but were no longer detected at 4 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). By contrast, the no-inflammatory CD163\u003csup\u003e+\u003c/sup\u003e macrophages, were evenly detected in all the experimental groups, exhibiting a homogenous distribution (Supplemental Fig.\u0026nbsp;1G, 1H).\u003c/p\u003e \u003cp\u003eThese data suggest that monocytes recruited to the injured heart differentiate into M1/CD68\u003csup\u003e+\u003c/sup\u003e macrophages in the early phase at 72h and 1-week post-intervention, while M2/CD163\u003csup\u003e+\u003c/sup\u003e macrophage levels remain relatively unchanged.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMonocyte subtypes transcriptome is enriched in genes participating in inflammation and pro-fibrotic pathways.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we sought to examine the transcriptome changes of monocytes subsets after I/R, performing genes profiling on sorted monocytes (CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e and CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e) from SHAM and I/R rats 24h post-intervention to analyze inflammatory genes in an early phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Principal Component Analysis (PCA) of the global transcriptome revealed pronounced segregation and well-defined clustering of the analyzed samples driven by both monocyte subtypes and experimental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The analysis identified 304 differently expressed genes in CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e monocytes, including 129 upregulated and 175 downregulated, while in CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e monocytes, 184 genes were differently expressed, with 68 upregulated and 116 downregulated, compared with analogous populations in SHAM operated rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Volcano plots illustrated these differentially expressed genes in both monocyte subsets, highlighting genes with high fold-changes and statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In addition, heatmap analysis indicated that gene expression profiles clustered reliably by cell type and condition (Supplemental Fig.\u0026nbsp;2A, 2B).\u003c/p\u003e \u003cp\u003eTo further understand the biological roles of the differentially expressed genes in activated monocyte subsets after I/R, we performed functional enrichment analysis using PANTHER classification system. The analysis revealed that both CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e and CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e monocytes were significantly enriched in pathways related to TGF- beta signaling and inflammation mediated by chemokine and cytokine signaling pathways. CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e monocytes also showed enrichment in angiogenesis, FGF and interleukin signaling pathways, among others (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eFurthermore, Gene Ontology (GO) enrichment analysis indicated that leukocyte regulation and extracellular matrix organization were enriched in CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e monocytes, both of which are closely linked to inflammatory responses (Supplemental Fig.\u0026nbsp;2C). In CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e, the analysis highlighted GO terms associated with the extrinsic apoptosis signaling pathway and chemokine production (Supplemental Fig.\u0026nbsp;2D). Finally, the GSEA (Gene Set Enrichment Analysis) further confirmed the existence of distinct functional gene expression profiles between the two monocyte subsets following I/R (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH), supporting the hypothesis that each subset may play a specialized role in the post-ischemic inflammatory response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe secretome of immune cells isolated from I/R rats induces changes in fibrotic and apoptotic gene expression.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven the observed changes in neutrophils and monocytes \u003cem\u003ein vivo\u003c/em\u003e in I/R rat models, we hypothesized that the secretome of these activated immune cells could directly modulate cardiac gene expression. Therefore, we exposed healthy rat cardiac fibroblasts and cardiomyocytes to a conditioned media collected from activated neutrophils and monocytes isolated from I/R rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Taking in consideration the transcriptomics results we focused on the potential role of these immune cell secretomes in modulating fibrotic and apoptotic processes after the ischemic injury. First, we confirmed that I/R induces cardiac fibrosis as soon as 1 week after the intervention, as assessed by Masson\u0026rsquo;s trichrome staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Second, we analyzed the expression of fibrotic genes \u003cem\u003eCol1\u003c/em\u003e, \u003cem\u003eCol3\u003c/em\u003e and \u003cem\u003eTgfβ1\u003c/em\u003e and apoptotic genes \u003cem\u003eApaf1, Bcl2\u003c/em\u003e and \u003cem\u003eCycs\u003c/em\u003e in cardiomyocytes and fibroblast treated with immune cells secretome. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD shows that the stimulation of cardiomyocytes with conditioned medium from monocytes isolated from I/R rats significantly increased \u003cem\u003eCol1\u003c/em\u003e and \u003cem\u003eCol3\u003c/em\u003e mRNA expression compared to SHAM controls, while neutrophil secretome had no significant effect on these gene expression. By contrast, only \u003cem\u003eTgfβ1\u003c/em\u003e expression was significantly increased in cardiac fibroblasts stimulated with I/R neutrophils conditioned medium whereas monocytes secretome had no effect in fibrotic genes expression.\u003c/p\u003e \u003cp\u003eMoreover, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE shows that the stimulation of cardiomyocytes with I/R monocytes conditioned medium trended to increase \u003cem\u003eCycs\u003c/em\u003e and \u003cem\u003eBcl2\u003c/em\u003e expression. In contrast, exposure to I/R neutrophil conditioned medium led to a significant reduction in Apaf1 gene level, without affecting \u003cem\u003eBcl2\u003c/em\u003e and \u003cem\u003eCycs\u003c/em\u003e expressions. In fibroblasts, I/R neutrophils supernatant significantly upregulated both \u003cem\u003eApaf1\u003c/em\u003e and \u003cem\u003eBcl2\u003c/em\u003e, whereas I/R monocytes conditioned medium do not affect their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These findings further validate the distinct and cell-type-specific influence of monocyte and neutrophil secretomes on fibrotic and apoptotic gene regulation in cardiac cells.\u003c/p\u003e \u003cp\u003eTo assess whether these findings can be confirmed in patient samples, we analyzed the expression of fibrotic and apoptotic genes in PBMCs from STEMI patients. Samples collected before (0h) and 6\u0026ndash;12h after revascularization were used to evaluate gene expression using a custom 75-gene Prime qPCR array. As shown in Supplemental Fig.\u0026nbsp;3A, PBMCs collected before revascularization (0h) exhibited a balanced pattern of up- and downregulated genes. However, 6\u0026ndash;12h post-revascularization, we observed a predominant upregulation of gene expression (52 out of 75 assessed genes, Supplemental Fig.\u0026nbsp;3B). Subsequently, using RT-qPCR we confirmed that \u003cem\u003eAKT2\u003c/em\u003e, \u003cem\u003eHMOX1\u003c/em\u003e, \u003cem\u003eMYC\u003c/em\u003e and \u003cem\u003eNFKB1\u003c/em\u003e were significantly upregulated in STEMI patients after the revascularization compared to controls, all of which are associated with apoptotic and fibrotic processes (Supplemental Fig.\u0026nbsp;3C).\u003c/p\u003e \u003cp\u003eThese results support the preclinical data and demonstrate that myocardial infarction induces widespread dysregulation of inflammation, fibrosis, and apoptosis-related genes in both animal model and human patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003emiRNAs overexpressed in inflammatory monocytes modify fibrosis and apoptosis-related genes expression.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eConsidering the previous results, we hypothesized that miRNAs expressed by the inflammatory monocytes could play a key role mediating the observed changes in gene expression. Therefore, we performed a miRNAs array analysis in sorted peripheral blood monocytes (CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e and CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e) from I/R rats. PCA analysis showed clear separation and clustering of the samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We found 935 differently expressed miRNAs in CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e and 669 miRNAs in CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Volcano plots and heatmaps illustrated these miRNAs expression profiles in both monocyte subsets from I/R rats compared to SHAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, Supplemental Fig.\u0026nbsp;4A).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing the datasets, we performed an \u003cem\u003ein-silico\u003c/em\u003e analysis to determine which miRNAs are predicted to modulate previously characterized fibrotic genes (\u003cem\u003eCol1\u003c/em\u003e, \u003cem\u003eCol3\u003c/em\u003e and \u003cem\u003eTgfβ1\u003c/em\u003e) and apoptotic genes (\u003cem\u003eApaf1, Bcl2\u003c/em\u003e and \u003cem\u003eCycs\u003c/em\u003e) in I/R rats. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, Supplemental Fig.\u0026nbsp;4B). The analysis revealed 9 common miRNAs: miR-16-5p, miR-18a-5p, miR-27a-3p, miR-29b-3p, miR-30b-5p, miR-30e-5p, miR-125a-5p, miR-194-5p and miR-222-3p, in both types of monocytes. Subsequent validation by RT-qPCR confirmed that miR-16-5p, miR-27a-3p, miR-29b-3p, miR-30b-5p, miR-30e-5p, miR-194-5p were significantly upregulated in CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003emonocytes from I/R compared to SHAM rats, but not miR-18a-5p, miR-125a-5p, and miR-222-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Conversely, the level of these miRNAs did not change in CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e monocytes except for miR-27a-3p (Supplemental Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further characterize the regulatory mechanisms mediated by miRNAs, we selected miR-16-5p, miR-27a-3p and miR-30b-5p to explore their impact on the expression of fibrotic and apoptotic genes in cardiac fibroblast and cardiomyocytes. Rat neonatal fibroblasts and ventricular myocytes (NRVM) were transfected with mimics of these miRNAs under control conditions and following simulated I/R. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Supplemental Fig.\u0026nbsp;6, I/R significantly increased \u003cem\u003eCol1\u003c/em\u003e and \u003cem\u003eCol3\u003c/em\u003e mRNA expression in both NRVM and fibroblasts. Interestingly, miR-16-5p and miR-30b-5p mimics significantly downregulated \u003cem\u003eCol1\u003c/em\u003e and \u003cem\u003eCol3\u003c/em\u003e expression in both cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), whereas miR-27a-3p mimic had no significant effect in NRVM (Supplemental Fig.\u0026nbsp;6). Notably, miR-30b-5p mimic upregulated \u003cem\u003eTGFB1\u003c/em\u003e expression in NRVM.\u003c/p\u003e \u003cp\u003eWe next examined the expression of apoptosis-related genes \u003cem\u003eApaf1\u003c/em\u003e, \u003cem\u003eBcl2\u003c/em\u003e, and \u003cem\u003eCycs\u003c/em\u003e under similar experimental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and Supplemental Fig.\u0026nbsp;6). I/R stimuli led to a slightly increase of \u003cem\u003eApaf1\u003c/em\u003e and decrease of \u003cem\u003eBcl2\u003c/em\u003e in NRVMs and fibroblast, while their transfections with miR-16-5p mimic (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) prevented I/R-induced \u003cem\u003eApaf1\u003c/em\u003e and \u003cem\u003eBcl2\u003c/em\u003e induced dysregulation in NRVM and/or fibroblasts. Conversely, miR-30b-5p mimic selectively downregulated \u003cem\u003eBcl2\u003c/em\u003e in fibroblasts but did not affect \u003cem\u003eApaf1\u003c/em\u003e expression in either cell type. Moreover, miR-27a mimic prevented I/R-induced \u003cem\u003eApaf1\u003c/em\u003e upregulation and \u003cem\u003eBcl2\u003c/em\u003e downregulation in either cell type (Supplemental Fig.\u0026nbsp;6). On the other hand, none of the tested miRNA mimics significantly modified \u003cem\u003eCycs\u003c/em\u003e gene expression in either fibroblasts or NRVM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB; Supplemental Fig.\u0026nbsp;6). These findings indicate that miR-16-5p, miR-27a-3p, and miR-30b-5p distinctly regulate the expression of \u003cem\u003eBcl2\u003c/em\u003e and \u003cem\u003eApaf1\u003c/em\u003e in cardiac cells, thereby influencing the apoptotic processes initiated by I/R injury.\u003c/p\u003e \u003cp\u003eIn summary, our data demonstrate that miRNAs overexpressed in CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003emonocytes from I/R rats, modulate the expression of fibrosis- and apoptosis-related genes in cardiac fibroblasts and cardiomyocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe inflammatory response following myocardial infarction plays a pivotal role in adverse cardiac remodeling with the infiltration and activation of neutrophils and monocytes populations driving fibrosis and cardiomyocyte apoptosis. However, the functional heterogeneity of monocyte subsets and the specific molecular mechanisms they regulate in this context, still require further comprehensive studies. Our work aimed to understand these functional and molecular diversity by isolating two types of rat monocytes after an I/R heart injury. We determined the differentially regulated gene expression profiles of these cell subtypes and analyzed their miRNAs as regulatory intermediates, implicated in inflammatory cardiac remodeling.\u003c/p\u003e \u003cp\u003eThe monocytosis in humans after an acute myocardial infarction (AMI) has been studied as adverse remodeling marker in some human data sets, as revascularized STEMI patients (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In this study we found significantly high numbers of neutrophils and monocyte subsets in STEMI patients after a PPCI, consistent with our previous study (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Furthermore, the analysis of pro-inflammatory genes expressed by PBMCs from STEMI patients, before and after the PPCI by prime-PCR showed that the expression of several genes changed as early as 6h to 12h after the revascularization during the inflammatory state. Pro-apoptotic genes as MYC and anti-apoptotic genes as AKT2, HMOX1 and NFKB1 were significantly upregulated in STEMI patients after PPCI. These increase suggest a molecular response toward cellular survival and stress adaptation in PBMCs. Actually, the induction of the AKT2 and NFKB1 pathways could reflects an enhanced anti-apoptotic signaling environment. These results align with a recent single-cell transcriptomic study, which demonstrated a systemic activation of the NF-KB pathway in some monocytes cell clusters following a STEMI in patients (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Concomitantly, the increased expression of HMOX1 points to a cytoprotective mechanism against the acute oxidative stress secondary to cardiac damage, possibly modifying myocardial fibroblast phenotype as indicated previously (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), while the elevation of MYC transcripts may indicate cellular activation and metabolic reprogramming in response to systemic inflammatory state after the injury (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Overall, these data indicate that PBMCs from STEMI patients undergo a rapid and coordinated transcriptional shift after PPCI, engaging both pro‑survival and stress‑response programs.\u003c/p\u003e \u003cp\u003eAfter I/R injury, it is known that monocytes and neutrophils are the first immune cells to infiltrate and 24-48h after the insult, monocytes subsets are the most abundant cell types in the myocardium (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Herein and using an I/R rat model we confirm that these monocytes-macrophages inflammatory populations persist for the first 72h-1 week after injury and subsequently decrease 4 weeks later, consistent with previous findings (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Rat inflammatory cells are presumed to share similar properties with mice and human monocytes; however, these populations are partially studied in rat and remain poorly characterized. Recently, new membrane-markers of rat peripheral blood inflammatory cells were recently described allowing their detection by FACs (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). To address this gap, we performed a microarray to characterize gene transcription of these monocyte subsets following an I/R damage. The transcriptomic analysis revealed significant gene dysregulation in both populations, mainly associated with cardiac remodeling signaling pathway, including inflammation, fibrosis, and apoptosis. These findings are consistent with the well-established effect of the ischemic injury and inflammatory cell infiltration in the heart, which trigger extensive apoptosis and fibrosis replacement of dead cells (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe plasticity of monocytes and macrophages is known to allow dynamic changes in their phenotype in response to microenvironmental signals (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Functional differences among monocyte and macrophages subpopulations have been related to their inflammatory profiles and responsiveness to pro-inflammatory stimuli (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), which significantly may influence the immune regulation in ischemic heart diseases (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Accordantly, our transcriptomics analysis showed differences between the two subtypes of monocytes CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e and in CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e after I/R stimulation, with a high percentage of variance reflecting extensive differential gene expression in each subset. Along the same line of research, a tissue single-cell RNA-seq study described that monocytes and macrophages are more diverse than expected (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and their diversity in blood could be associated to cardiovascular risk factors such as smoking and hyperlipidemia (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In our dataset, rat monocytes transcriptomic analysis showed well-differentiated cell populations after I/R damage, reflecting well-defined cell populations despite it is not a single-cell RNA-seq analysis. Moreover, monocytes have dynamic transcriptomes in thrombotic disease (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) and after myocardial infarction (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Therefore, while single-cell sequencing techniques offer unprecedent insights and high-resolution into complex biological systems (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), complementary functional studies are still essential to elucidate these dynamic processes. Hence, we used the secretome of monocytes and neutrophils collected from I/R rats as stimuli for cardiac cells isolated form control rats to mimic immune cell communication with cardiomyocytes and fibroblasts, and to analyze the functional impact of immune cells on I/R-evoked adverse cardiac remodeling. Our findings suggest that neutrophil -derived factors, rather than monocytes, are more likely modulators of apoptotic gene expression while monocytes secretome influences fibrosis, consistent with previous results (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Accordantly, others studies demonstrated that these pro-inflammatory cell populations are able to change fibroblast and cardiomyocyte gene expression by chemokines production (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), or other inflammatory cells derived molecules including non-coding RNAs (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, we focused on the role of miRNAs, since our previous studies have demonstrated that miRNAs are rapidly dysregulated after PPCI in STEMI patients serum (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), in PBMCs (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), and in I/R rats serum and cardiac tissue (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). However, their specific role in monocytes after I/R has not been previously explored. Interestingly, we identify several dysregulated miRNAs expressed in CD43\u003csup\u003ehigh\u003c/sup\u003e His48\u003csup\u003elow\u003c/sup\u003e monocytes compared with CD43\u003csup\u003elow\u003c/sup\u003e His48\u003csup\u003ehigh\u003c/sup\u003e cells, suggesting their differential sensibility to I/R. The \u003cem\u003ein-silico\u003c/em\u003e analysis identified a repertoire of miRNAs, targeting key genes implicated in fibrosis and apoptosis. Among the identified and validated miRNAs, miR-16-5p, miR-27a-3p, and miR-30b-5p are known to be key players in cardiovascular physiology, including fibrosis and apoptosis (\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Interestingly, the incubation of neonatal fibroblasts and cardiomyocytes with mimics of these miRNAs efficiently changed expression levels of fibrotic and apoptotic genes. Specifically, miR-16-5p mimic diminished the level of collagen genes both in cardiomyocytes and fibroblast consistent with its reported anti‑fibrotic effects (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Moreover, miR-16-5p likely balances the expression of pro- and anti- apoptotic genes by increasing \u003cem\u003eBCL2\u003c/em\u003e expression, widely known for its anti-apoptotic action (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), and decreasing \u003cem\u003eAPAF1\u003c/em\u003e expression, which has a recognized role in initiating apoptosis (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Notably, this miRNA also predicts the adverse cardiac remodeling in STEMI patients (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Concordantly with our results, the miR-30 family, including miR-30b-5p, is known for its anti-fibrotic properties, directly suppressing the expression of extracellular matrix components like collagens, alongside with its impact on apoptotic processes post-myocardial infarction (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Finally, despite that miR-27a-3p exerts context-dependent effects on both fibrosis and apoptosis (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), we observed an anti-apoptotic effect of this miRNA in neonatal cardiomyocytes through inhibition of \u003cem\u003eAPAF1\u003c/em\u003e. Altogether, these roles underscore the complex regulatory network through which miRNAs may regulate adverse cardiac remodeling.\u003c/p\u003e \u003cp\u003eIn fact, several studies have proposed miRNAs as therapeutic targets to treat deleterious ischemic effects in the heart (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), detrimental consequences of atherosclerosis (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), and to enhance cardio-protection after a myocardial I/R injury (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Understanding the regulatory role of miRNAs could provide deeper insights into cells communication and the pathogenesis underlying adverse cardiac remodeling.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur work highlights the pivotal role of immune cell-derived factors in cardiac remodeling after I/R. We demonstrated that myocardial injury induces distinct gene and miRNAs expression profiles within rat monocyte populations, reflecting their activated states, and further revealed that the secretome rich on miRNAs from these activated immune cells could directly modulate key fibrotic and apoptotic gene expression in cardiac fibroblasts and cardiomyocytes. Importantly, we identified specific miRNAs, namely miR-16-5p, miR-27a-3p, and miR-30b-5p, as regulators of these processes. These data were corroborated in humans, which showed a dysregulation of inflammation, fibrosis, and apoptosis-related genes in PBMCs from STEMI patients after the revascularization. Altogether, our findings underscore the translational relevance of immune cell-mediated gene, miRNAs regulation and communication with myocardial cells in adverse cardiac remodeling, suggesting novel targets for therapeutic interventions to mitigate cardiac remodeling and preventing progression to heart failure.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eI/R\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eischemia/reperfusion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eacute myocardial infarction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTEMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eST-segment elevation myocardial infarction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eCOL1\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecollagen type 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eCOL3\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecollagen type 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eTGFB1\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransforming growth factor beta 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eAPAF1\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eapoptotic protease activating factor-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eBCL2\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eB-cell lymphoma 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eCYCS\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecytochrome c somatic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eAKT2\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAKT Serine/Threonine Kinase 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eHMOX1\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHeme Oxygenase 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eMYC\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMYC Proto-Oncogene, BHLH Transcription Factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eNFKB1\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNuclear Factor Kappa B Subunit 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted following the principles published by the declaration of Helsinki and its modification or similar ethical standards. The study was authorized by the local Ethics Committee on Human Research at the University Hospital \u0026ldquo;Virgen del Rocio\u0026rdquo; of Seville (Approvals no. 2013PI/096, 2018/352, 2021/03 and FIS-ISG-2024-01).\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eSupplementary information\u003c/h2\u003e\n\u003cp\u003eThe online version contains supplementary material available at\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eDebora Falcon is supported by EMERGIA talent fellowship. This study was funded by \u0026ldquo;European Regional Development Fund; A way of making Europe,\u0026rdquo; and by the \u0026ldquo;European Union\u0026rdquo; and the Andalusia Government (grants numbers: ProyExcel_00530, PI-0034-2021, PI-0020-2024), the Institute of Carlos III (grant no. PI18/01197, PI23/01925), and the Spanish Ministry of Economy and Competitiveness (PID2022-136279NB-C22).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eE.B. and D.F. substantially and equally contributed to the acquisition, analysis and interpretation of all data in the manuscript. I.G.O. contributed to the acquisition and analysis of data. F.M.C. contributed to the analysis of gene expression data. G.B.E. contributed with the acquisition of human samples and analysis of experimental human data and provided fundings. I.V. contributed with advice assistance and funding. A.O.F. T.S. and R.D.T. contributed to the conception and design of the work, interpretation of data and provided fundings. T.S. and R.D.T wrote the original manuscript. All authors read and approved the submitted manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thanks to A. Guisado and F. Guerrero-Marquez for providing peripheral blood human samples. We acknowledge the support of R. March from the Genomic\u0026rsquo;s facility, Mar\u0026iacute;a Jos\u0026eacute; Castro from flow cytometry\u0026rsquo;s facility and Rocio Duran from the Histology\u0026rsquo;s facility at IBiS.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article and its supplementary files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLevine GN, Bates ER, Blankenship JC, Bailey SR, Bittl JA, Cercek B, et al. 2015 ACC/AHA/SCAI Focused Update on Primary Percutaneous Coronary Intervention for Patients with ST-Elevation Myocardial Infarction An Update of the 2011 ACCF/AHA/SCAI Guideline for Percutaneous Coronary Intervention and the 2013 ACCF/AHA Guideline for the Management of ST-Elevation Myocardial Infarction. 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Cell Death Discov. 2025;11(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa XR, Yan TM, Pan Y, Jiang ZH. Optimization of siRNA therapeutics targeting MIAT for cardioprotection in myocardial ischemia/reperfusion injury. Mol Ther Nucleic Acids. 2025;36(4):102747.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"I/R injury, myocardial infarction, Inflammatory monocytes, miRNAs, cardiac fibrosis","lastPublishedDoi":"10.21203/rs.3.rs-9448122/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9448122/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIschemia-reperfusion (I/R) injury in myocardial infarction with ST-segment elevation (STEMI) can lead to detrimental effects on the myocardium. Although primary percutaneous coronary intervention (PPCI) has significantly improved patient survival, some patients still develop adverse left ventricular remodeling, primarily due to interstitial fibrosis. The aim of this study was to investigate the role of pro-inflammatory cell populations and their released microRNAs (miRNAs) in regulating cardiac fibrosis and apoptosis in a rat model of I/R.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFlow cytometry was used to measure the levels of pro-inflammatory cell subsets. Pro-inflammatory neutrophils and monocytes were isolated from peripheral blood and subsequently used in an \u003cem\u003ein vitro\u003c/em\u003e co-culture secretome assay to stimulate cardiac fibroblasts and cardiomyocytes. Microarray analysis of genes and miRNAs was performed on the two types of sorted pro-inflammatory monocytes 24h post-I/R to identify the inflammatory mediators responsible for pro-fibrotic and apoptotic genetic changes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIncreased levels of neutrophils and monocytes, similar to those observed in humans, were detected following I/R, concomitant with the presence of M1 macrophages within rat cardiac tissue. The inflammatory secretome derived from these populations was found to induce pro-fibrotic and pro-apoptotic gene expression in cardiac fibroblasts and cardiomyocytes. Gene microarray analysis revealed significant differences in the transcriptome of rat monocyte types, consistent with findings in human mononuclear cells from STEMI patients post-revascularization. Moreover, miRNAs microarray analysis identified differences in the expression of miR-16, miR-27, miR-29, miR-30, and miR-194, which are associated with pro-apoptotic and fibrotic genes regulation, 24h after the I/R procedure. Furthermore, miRNAs mimics of some of these miRNAs changed the gene transcription in fibroblasts and cardiomyocytes.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThrough combined co-culture analysis and miRNAs microarray screening, we were able to identify miRNAs as inflammatory mediators that modulate gene expression in myocardial and non-myocardial cells after I/R injury. These identified miRNAs could be used as therapeutic targets for reducing fibrosis and apoptosis after an ischemic event.\u003c/p\u003e","manuscriptTitle":"Impact of pro-inflammatory monocyte subsets and their microRNAs regulation after an ischemia/reperfusion myocardial injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 10:54:46","doi":"10.21203/rs.3.rs-9448122/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-01T18:22:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-21T08:24:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-21T08:23:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2026-04-17T10:49:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"87e2a98f-7182-4904-88ae-3d41a2369edd","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"68","date":"2026-05-01T18:22:44+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T10:54:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 10:54:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9448122","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9448122","identity":"rs-9448122","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}