Animal procedures were approved by the institutional ethical and animal care committees according to guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes, enforced in Spanish law under Real Decreto 1201/2005 (GVA authorized procedures 2020/VSC/PEA/0123 for the model of angiogenesis in NOD/SCID mice and 2020/VSC/PEA/0122 for the experimental model of myocardial infarction in nude rats).

Neonatal rat cardiomyocytes (NRCM) were isolated as described (Armiñán et al., 2009) with some modifications. Briefly, rats aged 1 to 2 days were euthanized by decapitation, and the hearts were extracted, minced and incubated overnight in HBSS (without Ca and Mg) with 0.0125% trypsin (Sigma Aldrich) with gentle agitation at 4°C. Subsequently, the fragments were digested for 20 min at 37°C in an enzyme mixture (0.2% collagenase, 1 mg/mL DNAse) in Leibovitz L15 medium (all from Gibco-Invitrogen, Grand Island, NY, United States). Cell suspensions were collected and centrifuged at 90 × g for 5 min at room temperature and the cells were resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% horse serum, 20% M-199 Medium, 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from Gibco). The cells were then incubated for 1.5 h under standard culture conditions (37°C in 5% CO₂) to allow fibroblasts to attach to the culture plates. Then, the medium containing unattached cells was centrifugated and cardiomyocytes were pelleted and seeded in 0.1% gelatin-coated plates with DMEM, 4% horse serum, 17% M-199 Medium, 5% FBS, and 1% penicillin/streptomycin. Primary cultures of human umbilical vein endothelial cells (HUVEC; ATCC, Manassas, VA, United States) were grown in Vascular Cell Basal Medium supplemented with Endothelial Cell Growth Kit-VEGF (ATCC).

The immortal MSC-TERT line was used as previously described (Gómez-Ferrer et al., 2021). Donor cells were cultured in DMEM-low glucose supplemented with 1% penicillin/streptomycin and 10% FBS (Lonza, Basel, Switzerland). FBS was first ultracentrifuged at 100,000 g for 16 h to remove contaminant bovine EVs. Cells were incubated in a humidified atmosphere (37°C in 5% CO₂) with EVs-free serum. EVs were collected after 48 h using a serial ultracentrifugation protocol (Gonzalez-King et al., 2017). Briefly, supernatants were first centrifuged at 2,000g for 10 min, then at 10,000g for 30 min and filtered through a 0.22-μmm filter. EVs were pelleted by ultracentrifugation at 100,000g for 70 min at 4°C (Optima L-100 XP, Beckman Coulter, Pasadena, CA, United States), washed with PBS, and pelleted again as before. Pellets were resuspended in 100 μl of PBS. Isolated EVs (10 mμL) were used for protein quantification with the BCA Protein Assay Kit (Pierce, Thermo Scientific Inc., Rockford, IL, United States). Where indicated, cells were treated with EVs by direct addition to the culture medium to a final concentration of 30 μg/mL shortly before the initiation of the different experimental conditions.

To incorporate miR-4732-3p into EVs, the later were resuspended in an electroporation buffer after the last ultracentrifugation step of isolation and were incubated with an artificial mimic of the chosen miRNA (MISSION miRNA mimic, Merck) at a concentration of 40 nM. The suspension was loaded into a Gene Pulser/MicroPulser Electroporation Cuvette and treated with 3 pulses of 300 V with 5 s between each pulse. Subsequently, the transduced EVs were incubated at 4°C for 30 min. To eliminate the miRNA not loaded in the EVs, the volume was increased with PBS to 25 mL and the solution was ultracentrifuged as described. The final pellet was resuspended in 100 μL of PBS.

Transfection of miR-4732-3p mimic in vitro was assessed using Lipofectamine 2000 (Thermo Fisher Scientific) according to manufacturer’s instructions. Briefly, 100 μL of miR-4732-3p mimic 1 μg/μL was mixed with vigorous shaking for 30 s with 200 μL of Lipofectamine. The mixture was incubated for 30 min at room temperature.

Transfection of miR-4732-3p mimic in vivo was assessed using Maxsuppressor in vivo RNA-LANCEr-II lipidic reagent (Cosmo Bio Co., Ltd., Japan) following manufacturer’s instructions. Briefly, 8.5 μl of semi-dry Maxsuppressor in vivo RNA-LANCEr-II was mixed with 16 μl of miR-4732-3p mimic in a total volume of 25 μl of PBS to a final dose of 25 μg/heart in each rat. The mixture was sonicated and used fresh for in vivo surgical procedures.

Reverse transcription was performed with the miRCURY LNA Universal RT miRNA PCR Kit (Qiagen, Madrid, Spain). The miRNA was quantified using a Viia TM 7 Real System and the results were analyzed with QuantStudio Real-Time PCR Software (Applied Biosystems, Foster City, CA, United States).

Oxygen-glucose deprivation was induced on NRCM by culture with DMEM without glucose, glutamine, and phenol red (Thermo Fisher Scientific) in a chamber at 1.5% O₂.

Neonatal rat cardiomyocytes were seeded at 2 × 10⁵ cells/cm² in 1% gelatin-coated plates. After OGD, the cells were detached from the plastic with trypsin/EDTA, and were washed and resuspended in binding buffer with annexin-V-FITC and propidium iodide for 15 min. The percentage of annexin-positive cells was measured by flow cytometry in a FACS Canto II cytometer (BD Bioscience, San Jose, CA, United States).

Neonatal rat cardiomyocytes were seeded at 2 × 10⁵ cells/cm² in 1% gelatin-coated plates. After OGD, the supernatant was tested for lactate dehydrogenase using the Cytotoxicity Detection KitPLUS (LDH) (Roche, Indianapolis, IN, United States).

Hypoxia-treated cells were washed three times with PBS and incubated with CellRox-FITC^® (Thermo Fisher) at a dilution of 1/200 in DMEM with no FBS. After 20 min incubation at 37°C the cells were washed three times in PBS to eliminate the unconjugated stain and were detached with trypsin/EDTA. The fluorescence intensity of the staining was measured by flow cytometry.

To test the effect of OGD and EV treatment on cardiac contraction, we seeded cardiomyocytes at high confluence in gelatin-coated 6-well plates (5 × 10⁵ cells/well). After several days cardiomyocytes start to beat spontaneously. The number of beats per minute was measured using a Leica DM6000 inverted optical microscopy at 10 × magnification. Cells were treated with 30 μg/mL of EVs or transfected with 20 nM encapsulated miR-4732-3p using Lipofectamine as indicated above and were then subjected to OGD for 6 h, and each hour the beats were counted and normalized to non-treated cells.

Fibroblasts from neonatal rats were seeded in a 48-well plate at 2 × 10⁵ cells/well. Fibroblasts were treated the day before the analysis with 10 ng/mL of TGF-β to stimulate differentiation into myofibroblasts. Cells were then treated with 30 μg/mL of EVs or transfected with 20 nM miR-4732-3p using lipofectamine as indicated above. A straight line in the monolayer of fibroblast was created using a 20-μL pipette tip. Images were taken 24 h after the addition of treatments using a Leica DM600 inverted microscope at 4 × magnification. The area of the scratch wound was then measured using ImageJ.

Cardiac fibroblasts were cultured on glass coverslips at 2 × 10⁴ cell/glass in a 24-well plate. After treatment with TGFβ and EVs, cells were fixed in 4% paraformaldehyde for 10 min, washed three times with PBS and permeabilized with 0.1% Triton X-100 in PBS for 25 min. Cells were then washed with PBS and blocked with 10% FBS at room temperature for 30 min. Next, the cells were incubated with anti-actin, α-smooth muscle-Cy3 (Sigma-Aldrich) or anti-col1A (Cell Signaling Technology) at a concentration of 1/200 in a humidified incubator overnight. After washing in PBS with PBS, and in the case of col1A treated with the Alexa488 Donkey anti-rabbit (BD Bioscience) for 1 h, the cells were washed with PBS and counterstained with DAPI for 15 min and mounted using a drop of FluorSave (CalbioChem, San Diego, CA, United States). Images were captured with a Leica DM2500 fluorescence microscope.

Tubular-like formations were measured using a described assay (Gonzalez-King et al., 2017). HUVEC cells were counted and seeded at 1.5 × 10⁴ cells/well in a 96-well plate pre-coated with 50 μL of growth factor-reduced Matrigel^® (BD Bioscience). Cells were incubated in a starvation medium to simulate the conditions of the infarcted heart (DMEM no glucose, 1% FBS) for 6 h with 30 μg/mL of EVs from MSC-TERT cells or transfected with 20 nM miR4732-3p. Images were taken using a Leica DM600 microscope at 10 × magnification and were analyzed using the online software Wimasis WimTube (WimTube: Tube Formation Assay Image Analysis Solution. Release 4.0¹).

Angiogenesis was assayed in vivo by measuring blood vessel formation from subcutaneous tissue sprouted into a semi-solid gel of basement membrane containing the test sample, as described (Malinda, 2009). Growth factor-reduced Matrigel (350 μL) was mixed in liquid form at 4°C with 50 μL of EVs or 20 μg of encapsulated miRNA mimic (57 ng/μL). The Matrigel mix was injected subcutaneously into the flanks of 6–8-week-old athymic nude mice. After 14 days, mice were sacrificed and the Matrigel plugs were excised and fixed in 4% formaldehyde in PBS. Plugs were embedded in paraffin, sectioned, and immunostained with anti-CD31 antibodies and examined for growth of blood vessels. As a positive control, 400 μL of Matrigel containing 100 ng/mL fibroblast growth factor (FGF) and 20 U of heparin were injected, and as a negative control 400 μL of Matrigel containing saline vehicle were injected.

Data are represented as mean ± SD. Student’s t-test was used for unpaired samples in the comparison between groups and the Chi-Square test was used when contingency tables were analyzed. When the distribution was not normal the Mann-Whitney U test was used. Analyses were conducted with GraphPad Prism 8 software. Differences were considered statistically significant at p < 0.05 with a 95% confidence interval.

Extracellular vesicles are postulated to be effective for cardiac repair (Zhao et al., 2015; Forsberg et al., 2020; Nazari-Shafti et al., 2020). However, the complexity involved in producing homogeneous populations in sufficient quantity hampers their clinical use. To overcome this problem, we previously immortalized dental pulp-derived primary MSC cultures by overexpressing human telomerase enzyme (termed MSC-T) using a lentiviral vector that provided resistance to hygromycin and increased telomerase activity. MSC-T exhibit prolonged lifespan and maintain cell division long after equivalent unmodified MSCs reach replicative senescence (Gómez-Ferrer et al., 2021). We harvested EVs from conditioned media of MSC-T cells using ultracentrifugation as described (Gonzalez-King et al., 2017). EVs from MSC-T were characterized by western blotting, nanotracking analysis and electron microscopy. The presence of the tetraspanin markers ALIX, HSP70, TSG101, and CD9 confirmed the presence of EVs (Figure 1A). The concentration and size of these vesicles was determined by nanotracking analysis and revealed structures of 100–130 nm (Figure 1B). This was confirmed by electron microscopy, which revealed circular membranous structures of 100 nm (Figure 1C), and further analysis of tetraspanins by immunogold staining with anti-CD63 antibodies revealed the accumulation of gold nanoparticles in vesicular structures (Figure 1D). We next analyzed the EV cargo by RNA-seq analysis and we examined for miRNAs that were regulated by hypoxia in parental MSC-T. Among them, we identified miR-4732-3p (Figure 1E), which was also found in cardiac cells at different intracellular abundance. Analysis of absolute copy number of miR-4732-3p per 0.5 × 10⁶ seeded cells was 73.46 ± 119.49 copies in NRCM 434.45 ± 134.51 in human umbilical vein endothelial cells (EC) and 595.02 ± 159.38 in rat cardiac fibroblasts (cFib). When these cultures were independently subjected to oxygen/glucose deprivation (OGD), the levels of intracellular miR-4732-3p were measured by qPCR and normalized to miRNA levels in cells cultured in standard conditions. These levels were significantly upregulated in NRCM and EC but downregulated in cFib (Figure 1F), indicating that this miRNA might play a specific role in different cardiac populations.

Extracellular vesicles are known to mediate cardioprotection in therapeutic interventions designed to mitigate I/R cardiac injury, and it is believed that EV miRNAs play a pivot role in this process (Barile et al., 2017; Zhang et al., 2017; Wu et al., 2020). To analyze the potential therapeutic effect of miR-4732-3p, we first electroporated EVs with a miR-4732-3p mimic to increase its concentration in EV particles (Figure 2A). Of note, electroporation did not affect the concentration or size of the EVs (Supplementary Figure 1) and they remained suitable for functional studies although the loss of miRNA cargo due to electroporation process is unknown. As an alternative to electroporation, synthetic miR-4732-3p was formulated into liposomes as a delivery system before transfection. The anti-apoptotic effect of EVs, with or without electroporated miRNA-4732-3p, and also miR-4732-3p liposome complexes, was assessed in NRCMs cultured under OGD conditions. We found that the number of apoptotic NRCMs was significantly reduced to a similar degree by the addition of native EVs or EVs previously electroporated with the miR-4732-3p mimic. Interestingly, a similar level of cardioprotection was achieved with liposomal miR-4732-3p (Figure 2B). Of note the control electroporation of EVs with saline reduced their anti-apoptosis action, likely due to the loss of vesicle integrity. In the context of electroporation, EVs that incorporated miRNA-4732-3p showed improved anti-apoptotic effects over electroporated vesicles with no miRNA (EVs_saline) or scramble miRNA (not shown). The finding that transfected miR-4732-3p could induce a similar reduction in apoptosis to addition of EVs underlines the potential role of this miRNA in cardioprotective mechanisms.

Analysis of reactive oxygen species production and LDH activity in NRCMs treated with either EVs or transfected with miR-4732-3p mimic before OGD revealed similar levels of cardioprotection (Figures 2C,D). While the amount of miR-472-3p delivered by EVs is much lower than the transfected amount, it is likely that other miRNAs in EVs play a role in the cardioprotection. To analyze the global response of miR-4732-3p in comparison with EVs, we tested the contractile profile of cardiomyocytes during ischemia. When we added EVs to NRCM cultures before the OGD episode, we observed that cells maintained contraction capacity in comparison with untreated NRCMs (Figure 2E). NRCMs transfected with miR-4732-3p also showed an improvement in their contraction capacity but the effect was not as pronounced (Figure 2E). To further explore this mechanism, we analyzed the levels of connexin 43 and caspase 3 by western blotting. Results showed that whereas cleaved caspase 3 was less abundant in NRCMs treated with EVs or miR-4732-3p suggesting a possible interaction of the miRNA with this pathway. However, further experiments are needed to confirm this interaction like a Cas3 inhibition assay. EVs exerted a greater upregulation of connexin 43, which could explain why EVs were more effective in preserving cell beating after cardiac injury (Figure 2F).

We next sought to analyze the response of cardiac fibroblasts (cFib) to miR-4732-3p since these cells play an active role in myocardial remodeling after injury. Among the putative target genes of miR-4732-3p are FGFs, angiotensin II and members of the TGFβ family including TGFβ, TGFβR1/3 and SMAD2/4² (not shown). To test the anti-fibrotic effect of miR-4732-3p, we added EVs or the miR-4732-3p mimic to fibroblasts treated with TGFβ using a scratch assay. Results confirmed that the migratory capacity was lower in fibroblasts transfected with miRNA-4732-3p and treated with TGFβ than in control TGFβ-treated fibroblasts. By contrast, EV treatment failed to affect the pro-migratory activity of TGFβ (Figure 3).

A major consequence of ischemic damage is the formation of a scar in the infarcted area derived from the differentiation of fibroblasts to myofibroblasts (Shinde and Frangogiannis, 2014). After myocardial infarction, a cytokine storm induces myofibroblast differentiation via angiotensin II and TGFβ, among others (Frangogiannis, 2015), Myofibroblasts have a greater contractile and invasion capacity and also a higher production of extracellular matrix components; hence, the excessive differentiation to myofibroblasts results in scar formation with no contraction capacity. To evaluate the effect of miR-4732-3p on myofibroblast induction, we treated cardiac fibroblasts with 10 ng/mL TGFβ to simulate differentiation. Myofibroblast differentiation was characterized by an increase in the expression of smooth muscle actin (α-SMA) and increased production of extracellular matrix proteins like collagen IA (col I-α1) (Figure 4). Although both EVs and transfection of miR-4732-3p led to a reduction in α-SMA expression, only miR-4732-3p treatment reduced collagen production. Taken together, the results suggest that miR-4732-3p inhibits myofibroblast differentiation and the production of extracellular matrix, which might control fibrosis in the ischemic area.

We tested the capacity of miR-4732-3p to induce the formation of new vasculature in vitro. We did not use OGD treatment since in those conditions tube formation was drastically impaired. Instead, HUVEC were seeded in DMEM without glucose and glutamine (Thermo Fisher Scientific) in growth factor-reduced Matrigel (GD condition). Results showed a significant increase in the formation of tubular structures from HUVEC transfected with the miR-4732-3p compared with untreated cells (Figure 5A). Contrastingly, cells treated with EVs showed no significant increase in vascular structures at the doses analyzed. We next sought to test the angiogenic potential of miR-4732-3p in vivo. Nude mice were injected subcutaneously with a Matrigel plug containing either saline, FGF (as a positive control), EVs or miR-4732-3p (Figure 5B). Plugs were removed 15 days later and were embedded in paraffin for histological analysis (Figure 5C). Individual pugs are shown in Supplementary Figure 2. Quantification of angiogenesis was performed by measuring plug-infiltrating vessels using anti-CD31 staining. The results showed a potent angiogenic effect in mice treated with miR-4732-3p in comparison to EVs and control conditions (Figures 5D,E). Overall, these findings indicate that miR-4732-3p induces angiogenesis both under stress and in physiological conditions.

The ability of EVs derived from MSC isolated from different origins has been extensively reported and in most cases therapeutic effects have been attributed to miRNA cargo components (Wang et al., 2017; Luther et al., 2018; Zhu et al., 2018; Mao et al., 2019; Huang et al., 2020; Peng et al., 2020; Xu et al., 2020).

We tested whether the administration of miR-4732-3p could recapitulate this effect by encapsulating it in liposomes before intramyocardial implantation shortly after permanent LAD ligation in nude rats. Results were compared to saline intramyocardial injections. Four weeks after transplantation, cardiac function parameters were measured to assess the degree of functional recovery. The miR-4732-3p group displayed a significant recovery of systolic function as calculated by the percentage of fractional shortening (FS) and fractional area change (FAC) (Figure 6A). Treatment with miR-4732-3p also significantly reduced the area of fibrous scar tissue (Figure 6B). These in vivo data support the hypothesis that miR-4732-3p can induce cardioprotection against cardiac ischemia.