Extracellular vesicles from anoxia preconditioned mesenchymal stem cells alleviate myocardial ischemia/reperfusion injury

Characterization of ADSCs and ADSC-derived EVs

Successful isolation of ADSCs from mouse adipose tissue was verified through flow cytometry, which demonstrated positive expression (>95%) of the cell surface markers CD29, CD45, and CD90 and low/negative expression of CD34, CD105, and CD106 (Figure 1A). Subsequently, ADSC-derived EVs (ADSC-EVs) were obtained through sequential centrifugation and characterized using both a Nanosight device and transmission electron microscopy (TEM). These analyses indicated that the collected vesicles were mainly exosomes, with a median average size of 115 nm (range: 50-150 nm) (Figure 1C–1E). The isolated vesicles expressed three EV-associated markers, i.e. CD9, CD63, and tumor susceptibility gene 101 (TSG101) and were negative for calnexin (Figure 1D). Internalization of PKH67-labeled EVs by cultured neonatal mouse cardiomyocytes was confirmed by fluorescence microscopy (Figure 1F).

Figure 1. Characterization of mouse ADSCs and ADSC-derived EVs. (A) Analysis of cell surface markers (CD29, CD45, and CD90) in isolated mouse ADSCs; CD34, CD105, and CD106 were used as negative controls. (B) The purity of neonatal mouse cardiomyocytes was estimated to be higher than 95% based on α-actinin staining results. (C) TEM characterization of EVs. (D) Western blot examination of EV markers. Calnexin was used as negative control. (E) NanoSight’s light scattering intensity-based size distribution of isolated EVs. (F) EV tracer (PKH67) assay demonstrating successful EV internalization by cardiomyocytes.

ADSC-EVs reduce pyroptosis and apoptosis in cardiomyocytes subjected to anoxia/reoxygenation

To examine whether ADSC-EVs can prevent or attenuate MIRI-induced pyroptosis in cardiomyocytes, pyroptosis markers were detected by western blotting in cultured neonatal mouse cardiomyocytes which were subjected to anoxia/reoxygenation (AR) and were exposed to EVs derived from normoxic ADSCs (NC-EVs) or to EVs derived from anoxia-preconditioned ADSCs (Int-EVs) 2h prior to AR. As shown in Figure 2A, 2B, exposure to NC-EVs decreased the expression of both TXNIP and cleaved-caspase-1 (TXNIP, p=0.0007; cleaved-caspase1, p=0.0002; compared to control AR). However, the downregulation of the two pyroptosis markers was significantly higher after exposure to Int-EVs (TXNIP, p=0.0194; cleaved-caspase-1, p=0.0007; compared to NC-EVs).

Figure 2. Analysis of pyroptosis and apoptosis markers in EV-treated, AR-exposed cardiomyocytes. (A, B) Western blot analysis of TXNIP and cleaved-caspase-1 in AR-exposed cardiomyocytes incubated with EVs derived from normoxic (NC-EVs) or anoxic (Int-EVs) ADSCs. The NC-EVs group was compared with the control AR group and the Int-EVs group was compared with the NC-EVs group (*P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, n = 4). (C, D) Western blot analysis of BAX, cleaved-caspase-3, GATA4, and Bcl-2 in cardiomyocytes treated with AR. Comparisons were made between NC-EVs and AR and between Int-EVs and NC-EVs groups (*P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, n = 4). (E, F) Apoptosis detection in AR-exposed cardiomyocytes (Annexin V/PI assay). The NC-EVs group was compared with the AR group and the Int-EVs group was compared with the NC-EVs group (*P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, n = 4). (G, H) Relative expression of cleaved GSDMD and total GSDMD in cardiomyocytes and RAW264.7 cells treated with or without AR (*P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, compared with the control CM group; n = 4); (#P < 0.05, ##P < 0.01,###P < 0.001, #### P < 0.0001, compared with the CM + AR group; n = 4); (&P < 0.05, &&P < 0.01, &&& P < 0.001, &&&&P < 0.0001, compared with the RAW264.7 group; n = 4).

Active caspase-1 can cleave and deactivate GATA4, a key transcription factor regulating cardiac development and cardiomyocyte survival [8]. To evaluate whether ADSC-EVs can prevent both GATA4 downregulation and apoptosis in AR-treated cardiomyocytes, we conducted western blotting and Annexin V/PI assays following the manufacturer's instructions. As shown in Figure 2C, 2D, exposure to NC-EVs decreased the expression of cleaved-caspase-3 (a downstream target of cleaved caspase-1) and upregulated both GATA4 and the Bcl-2/Bax ratio (GATA4, p=0.036; Bcl-2/Bax, p=0.0007; cleaved-caspase-3, p=0.0058; compared to AR). Once again, however, the observed changes were significantly larger in Int-EV-treated cells (GATA4, p=0.0106; Bcl-2/Bax, p=0.0022; cleaved-caspase-3, p=0.0003; compared to NC-EVs). Meanwhile, as shown in Figure 2E, 2F, Annexin V-PI assays demonstrated a reduced rate of apoptosis after cardiomyocyte exposure to either Int-EVs or NC-EVs, with Int-EVs eliciting in turn a larger anti-apoptotic effect (NC-EVs vs AR, p=0.0002; Int-EVs vs NC-EVs, p=0.0143).

GSDMD expression in mouse cardiomyocytes is low and unaffected by AR

Caspase-1 mediated cleavage of GSDMD, a pore-forming protein, is a key event in the onset of pyroptosis triggered by the TXNIP/NLRP3 inflammasome pathway. Since AR-mediated TXNIP activation in cardiomyocytes fails to activate pyroptosis, we investigated the effect of AR on GSDMD expression in both mouse neonatal cardiomyocytes and myeloid RAW264.7 cells, which are known to express high GSDMD levels. As shown in Figure 2G, 2H, no differences in cleaved GSDMD expression were found between control (CM) and AR-exposed (CM + AR) cardiomyocytes (p=0.991). Meanwhile, stimulated with AR, expression of cleaved GSDMD in RAW264.7+AR group was significantly higher than other comparisons (p<0.0001 for all comparisons). In addition, total GSDMD expression was found to be much higher in RAW264.7 cells than in cardiomyocytes (p<0.0001).

ADSC-EVs ameliorate myocardial damage in a mouse model of MIRI

To evaluate whether ADSC-EVs can protect against MIRI-induced myocardial damage in vivo, cardiac infarct size (IS) and area at risk (AAR) were assessed in mice intravenously injected with ADSC-EVs before MIRI surgery. As shown in Figure 3B, AAR/LV values were similar between the sham, IR (MIRI), and IR + NC-EVs groups (re-ligating left coronary artery prior to Evens blue stain to calculate AAR/LV values). In contrast, the NC-EVs and Int-EVs groups exhibited significantly reduced post-MIRI IS compared to the IR group, with the Int-EVs group showing again the most significant effect (NC- EVs vs IR; p=0.0007; Int- EVs vs NC-EVs; p=0.0028).

Figure 3. Cardioprotective effects of ADSC-derived EVs in a mouse model of MIRI. (A) Apoptosis (TUNEL) assay in mouse myocardial sections obtained 12h after reperfusion. The NC-EVs group was compared with the IR group and the Int-EVs group was compared with the NC-EVs group (Image below: 400× magnification; *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, n = 6). (B) Assessment of infarct size (IS) and area at risk (AAR) in mice subjected to MIRI. Results were compared between the NC-EVs and the IR groups and between the Int-EVs and the NC-EVs groups (10× magnification; IR: ischemia reperfusion; TTC: triphenyltetrazolium chloride; AAR: *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, n = 6). (C, D) Western blot analysis of TXNIP, cleaved-caspase-1, and NLRP3 in infarcted mouse myocardial tissue. Data were compared between the NC-EVs and the IR groups and between the Int-EVs and the NC-EVs groups (*P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, n = 4). (E, F) Western blot analysis of BAX, cleaved-caspase-3, GATA4, and Bcl-2 expression in infarcted mouse myocardial tissue. Data were compared between the NC-EVs and the IR groups and between the Int-EVs and the NC-EVs groups (*P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, n = 4). (G, H) Cardiac function assessment. Echocardiography was used to examine EF and FS immediately after sham or MIRI surgery (baseline) and on the 3^rd and 7^th day after reperfusion (#P < 0.05, ##P < 0.01, ### P < 0.001, #### P < 0.0001, NC-EVs vs IR; n = 7). (*P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, Int-EVs vs NC-EVs; n = 7).

In vivo infusion of ADSC-EVs inhibits MIRI-induced cardiomyocyte death

To confirm that ADSC-EVs attenuate MIRI by promoting cardiomyocyte survival, apoptosis and pyroptosis were examined in myocardial tissue ex-vivo following intravenous infusion of ADSC-EVs and MIRI surgery in mice. TUNEL assay results showed that administration of NC-EVs reduced the rate of apoptosis in the myocardium (NC-EVs vs IR; p=0.0003), an effect that was, in turn, significantly enhanced after injection of Int-EVs (Int-EVs vs NC-EVs, p=0.0141) (Figure 3A). As shown in Figure 3E and 3F, these pro-survival effects were paralleled by decreased expression of cleaved-caspase-3, upregulated expression of GATA4, and increased Bcl-2/Bax ratio in myocardial cells (NC-EVs vs IR: GATA4, p=0.0026; Bcl-2/Bax, p=0.0001; cleaved-caspase-3, p=0.0011; Int-EVs vs NC-EVs: GATA4, p=0.0242; Bcl-2/Bax, p=0.027; cleaved-caspase-3, p=0.0006).

To evaluate whether ADSC-EVs could also inhibit MIRI-related pyroptosis of cardiac cells, pyroptosis pathway markers were detected in the excised hearts. As shown in Figure 3C, 3D, the expression of both TXNIP and cleaved-caspase-1 decreased upon pre-treatment with NC-EVs (TXNIP, p=0.009; cleaved-caspase1, p=0.0078; compared to IR). Of note, further reductions in TXNIP, cleaved-caspase-1, and NLRP3 levels were detected in samples from Int-EV-treated mice (TXNIP, p=0.0042; NLRP3, p=0.0341; cleaved-caspase1, p=0.0287; compared to NC-EVs).

ADSC-EVs improve cardiac function after MIRI

Echocardiography on days 3 and 7 post-MIRI revealed that ejection fraction (EF) and left ventricular fractional shortening (LVFS) were significantly improved in mice that received ADSC-EVs (Figure 3G, 3H), and especially Int-EVs (EF day 3: NC-EVs vs IR, p=0.020; Int-EVs vs NC-EVs, p=0.036; FS day 3: NC-EVs vs IR, p=0.033; Int-EVs vs NC-EVs, p=0.015; EF day 7: NC-EVs vs IR, p=0.022; Int-EVs vs NC-EVs, p=0.017; FS day 7: NC-EVs vs IR, p=0.058; Int-EVs vs NC-EVs, p=0.014).

Anoxic preconditioning upregulates miR-224-5p, a potential TXNIP regulator, in ADSC-EVs

To explore the mechanism underlying the higher inhibitory efficacy of Int-EVs on AR-mediated cardiomyocyte cell death, microRNA (miRNA) sequencing was performed on NC-EVs and Int-EVs. A total of 41 miRNAs were upregulated in Int-EVs compared to NC-EVs (Supplementary Table 1). Among those, 10 were predicted to associate with TXNIP by the TargetScan and miRanda algorithms in The Encyclopedia of RNA Interactomes (ENCORI) database. Differential expression of these miRNAs was confirmed by real-time PCR (Figure 4A, 4B). Among the 10 upregulated miRNAs predicted to associate with TXNIP, miR-224-5p was selected for further study because its upregulation was the most significant. Dual-luciferase reporter assay results showed that the fluorescence ratio was significantly reduced from HEK 293T cells upon co-transfection with miR-224-5p mimics and TXNIP WT-3’ UTR, compared to the NC and mutant 3’ UTR controls (p=0.0011) (Figure 4C, 4D). These results indicated that miR-224-5p inhibits the translation of TXNIP by targeting the 3’ UTR region of its mRNA.

Figure 4. Anoxia-induced, EV-associated miR-224-5p inhibits I/R-mediated apoptosis in cardiomyocytes by downregulating TXNIP. (A) MicroRNA sequencing analysis in ADSC-EVs. A total of 41 miRNAs were upregulated in Int-EVs compared to NC-EVs. Ten of them (shown in the heatmap) were predicted to associate with TXNIP. (B) Validation of differential EV-associated miRNA expression through qRT-PCR. U6 snRNA served as internal reference (*P < 0.05, compared with NC-EVs; n=3). (C) Dual-luciferase reporter assay. HEK 293 T cells were co-transfected with miR-224-5p mimics and PGL3 luciferase reporter plasmids containing wild-type or mutated TXNIP 3′UTR. An NC-TXNIP 3′UTR served as control. (D) Dual-luciferase reporter assay analysis (*P < 0.05, miR-224-5p + TXNIP WT 3′UTR vs mutated- and NC-3’UTR groups; n=3). (E) Analysis of TXNIP and miR224-5p expression in AR-treated mouse cardiomyocytes transfected with a miR224-5p inhibitor and/or TXNIP-targeting siRNAs (TXNIP-RNAi). (F) Statistical analysis of data from experiments like those shown in (E); (&P < 0.05, &&P < 0.01, &&& P < 0.001, &&&&P < 0.0001, compared with the control group; n = 3; *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, compared with the AR group; n = 3; #P < 0.05, ##P < 0.01, ### P < 0.001, #### P < 0.0001, compared with the AR + inhibitor group; n = 3). (G) Representative images of Annexin V/PI-stained cardiomyocytes transfected with a miR224-5p inhibitor and/or TXNIP-targeting siRNAs prior to AR. a) NC: non-AR control; b) AR; c) Inhibitor-NC + AR: miR224-5p inhibitor (negative control) pre-treatment followed by AR; d) TXNIP-RNAi + AR: TXNIP-RNAi pre-treatment followed by AR; e) Inhibitor + AR: miR224-5p inhibitor pre-treatment followed by AR; f) Inhibitor + TXNIP-RNAi + AR: miR224-5p inhibitor plus TXNIP-RNAi pre-treatment followed by AR. (H) Statistical analysis of Annexin V/PI staining experiments. (&P < 0.05, &&P < 0.01, &&& P < 0.001, &&&&P < 0.0001, compared to control; n = 4; *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001, compared to AR; n = 4; #P < 0.05, ##P < 0.01, ### P < 0.001, #### P < 0.0001, compared to miR224-5p inhibitor + AR; n = 4).

miR-224-5p ameliorates AR-induced cardiomyocyte apoptosis by targeting TXNIP

To assess whether miR-224-5p can attenuate AR-induced apoptosis in cardiomyocytes by inhibiting TXNIP, western blotting and Annexin/V-PI assays were performed on AR-exposed neonatal mouse cardiomyocytes pre-treated with a miR-224-5p inhibitor or a TXNIP-targeted siRNA (TXNIP-RNAi) to silence TXNIP expression. As shown in Figure 4E, 4F, transfection with the miR-224-5p inhibitor decreased miR-224-5p and increased TXNIP expression relative to untransfected control (AR) cells (p=0.022). AnnexinV/PI staining results (Figure 4G, 4H) indicated, as expected, significant induction of apoptosis in AR-treated cardiomyocytes, compared to non-AR control (CON) cells (p<0.0001). Of note, this effect was significantly enhanced by inhibition of miR-224-5p (p=0.0115, compared to AR). In contrast, inhibition of TXNIP in cardiomyocytes undergoing AR significantly reduced the apoptosis rate (p=0.0025, compared to AR). Importantly, the pro-apoptotic effect elicited by miR-224-5p suppression could be reversed by co-transfection with TXNIP-RNAi (p <0.0001). These results demonstrated that miR-224-5p inhibits apoptosis of cardiomyocytes undergoing AR by targeting TXNIP.

MIRI mouse model

All animal care and procedures were approved by the Shanghai Ninth People’s Hospital Institutional Ethics Committee (SH9H-2017-A39-1) and performed in accordance with the guidelines of the Directive 2010/63/EU of the European Parliament. Six-week-old male, specific-pathogen-free (SPF) wild-type (WT) C57BL/6 mice were purchased from the Experimental Animal Center of Shanghai Ninth People’s Hospital. Animals were fed with standard mouse chow and water ad libitum under SPF conditions (20-24° C; 50-60% humidity). All invasive procedures were performed under anesthesia, using first an anesthesia box with 3% isoflurane (Sinopharm Chemical Reagent Co. Ltd, Shanghai, China) for 1 min and administering thereafter 1.5-2% isoflurane through an anaesthetic mask. To minimize experimental variability, all animal procedures (MIRI, Evans blue/TTC stain) were performed by Prof. Erhe Gao. Mice were euthanized by carbon dioxide inhalation.

A total of 80 healthy WT C57BL/6 male mice (eight weeks old, ~24 g) were used for experiments. The MIRI model was generated as described in detail in Gao et al. [43] by ligating the left coronary artery (LCA) for 30 min with a slipknot, then releasing it to induce reperfusion. Successful MIRI was confirmed on the basis of dynamic electrocardiograph (ECG) changes (ST-segment elevation). Sham-operated mice, used as control, underwent the same procedure, with the exception that the knot on the LCA was left untied.

Transthoracic echocardiography

Echocardiography was performed to assess ejection fraction (EF) and fractional shortening (FS) under 1.5-2% isoflurane anesthesia immediately after MIRI surgery (baseline) and on the 3^rd and 7^th days after reperfusion using a Vevo 770 high-resolution imaging system.

Evaluation of area at risk and infarct size

After reperfusion for 4 h, the chest wall was reopened under 1.5-2% isoflurane anesthesia to expose the heart. The LCA was then re-ligated and the distal artery nipped, and 1% Evans Blue was injected through the ascending aorta until the non-infarction zone turned blue. The heart was harvested, washed in saline, and sliced horizontally into five pieces, from the left ventricular apex until the ligation site. All tissue pieces were incubated in 1% TTC for 20 min at 37° C. Image Pro Plus 6.0 was used to calculate infarct area and area at risk (AAR). AAR/left ventricle area (LV) ×100% and infarct size (IS) /AAR×100% were calculated.

Isolation and culture of mouse adipose-derived stem cells

ADSCs were isolated from adipose tissue from C57BL/6 mice as previously described [44, 45]. In brief, adipose tissue isolated from the proximal limb was digested by 0.075% collagenase IV (Cat. 9001-12-1; Sigma, USA) for 60 min at 37° C in a mechanical horizontal rotator at a speed of 180 rpm. Collagenase IV was dissolved in complete medium containing DMEM/F12 (Cat. SH30023.01; Hyclone, USA) and 10 % fetal bovine serum (FBS; Cat. ST303302, PAN, Germany). After digestion, the samples were centrifugated at 200×g for 10 min and the cell pellets resuspended in complete medium (DMEM/F12 and 10% EV-free serum). The medium was then placed on 10 cm culture dishes in an incubator at 37° C in a 5% CO₂ atmosphere. The extraction methods were based on previous references [44, 45] and remained undisturbed for three days prior to collection of EVs. Characterization of ADSCs was performed by flow cytometry analysis of CD29, CD45, and CD90 (positive cell surface markers) and CD34, CD105, and CD106 (negative controls).

Isolation and culture of neonatal mouse cardiomyocytes

Cardiomyocytes were isolated from 1-2 day-old C57BL/6 mice as described previously [46]. In brief, mice were disinfected with a 75% ethanol solution and hearts quickly extracted and digested in 0.125% trypsin diluted with PBS at 4° C overnight. Following centrifugation at 200×g for 5 min, the cell pellet was resuspended and cultured in DMEM containing 10% FBS at 37° C in a 5% CO₂ atmosphere for 1.5 h to allow for fibroblast attachment. Culture supernatants containing cardiomyocytes were then plated in 0.1% gelatin-coated dishes at a density of 1 × 10⁶ cells/mL. The purity of isolated cardiomyocytes was estimated by α-actinin staining.

In vitro cardiomyocyte anoxia/reoxygenation model

An in vitro model of mouse cardiomyocyte anoxia/reoxygenation (AR) was established by incubating cells in oxygen-free, low-glucose DMEM in a controlled atmosphere (95% N2 and 5% CO₂) for 2 h and reinstating afterwards normal oxygen levels and standard DMEM for 1 h.

Anoxic preconditioning of ADSCs

ADSCs (1-2×10⁶ cells/100-mm cell-culture dish) were seeded in EV-free complete medium for 24 h. The medium was then exchanged for DMEM/F12 previously incubated with 100% N2 overnight to remove oxygen. Anoxic preconditioning was performed by exposing the cells to 5 cycles of anoxia (60 min in oxygen-free medium) with intermittent reoxygenation (30 min in normal DMEM/F12) in an anoxic chamber (Forma-1025 Anaerobic System, Thermo Fisher Scientific, USA). ADSCs were next incubated with serum-free medium in a normal incubation environment (21% O₂, 5% CO₂) and the supernatant was collected after 24 h.

Isolation and characterization of EVs

EVs were isolated from cultured ADSCs (exposed or not to anoxic preconditioning) by differential centrifugation in conditioned media. In brief, the cell culture supernatant was centrifuged at 2,000×g for 30 min at 4° C to remove cell debris. The supernatant was then collected and centrifuged at 100,000×g for 70 min to precipitate EVs. The supernatant was discarded to remove contaminating proteins and EVs were resuspended in PBS and centrifuged at 100,000×g for 70 min. Aliquots of the EV precipitates were preserved at - 80° C to avoid repeated freeze/thaw cycles. EVs derived from non-preconditioned ADSCs are herein referred to as NC-EVs, whereas those derived from anoxia-preconditioned ADSCs are termed Int-EVs. EV size distribution and concentration were verified by a NanoSight NS300 instrument (Malvern Instruments, Malvern, UK) and EV morphology defined using transmission electron microscopy (TEM). Expression of the EV markers TSG101, CD9 (positive controls), and calnexin (negative control) was assessed by western blot [47].

EV injections

Mice were randomly divided into 4 groups (n =20 mice per group): 1) sham (no MIRI; control); 2) MIRI; 3) NC-EVs (NC-EVs plus MIRI); and 4) Int-EVs (Int-EVs plus MIRI). On groups 3 and 4, EVs were administered at a dose of 10 μg per gram of body weight through tail vein injection prior to MIRI surgery.

Analysis of EV uptake by cardiomyocytes

EVs and mouse cardiomyocytes were labeled with PKH67 (Cat. MINI67, Sigma-Aldrich, USA) and phalloidin (Cat. A12379s, ThermoFisher Scientific, USA), respectively, according to the manufacturers’ protocols. A total of 5 μL of PKH67-stained EVs was added to each cardiomyocyte culture, followed by a 2-h incubation to allow internalization by the cells. After washing twice with PBS, slides were fixed in 4% paraformaldehyde and cell nuclei were stained with DAPI. Fluorescent images were collected using an inverted microscope (BX63, Olympus, Japan) at 630x magnification.

Cell transfection

293T cells were transfected with miR-224-5p mimics (Ribobio, Shanghai, China), Dual-Luciferase(DLU)-plasmid DNA using Lipofectamine 3000 transfection reagent (Cat. 11668019, Invitrogen, USA) following the manufacturer’s protocol. Mouse cardiomyocytes were transfected with a miR-224-5p inhibitor (RiboBio, Guangzhou, China) and small interfering RNAs targeting TXNIP (TXNIP-RNAi; RiboBio, Guangzhou, China) using RFect^PM transfection reagent (Cat.11011, BAIDAI, China) following the manufacturer’s protocol. Experiments were performed 24 h after transfection.

Alpha-actinin immunofluorescence

Mouse cardiomyocytes cultured on confocal plates were fixed in 4% paraformaldehyde for 15 min, washed with PBS 3 times, and permeabilized with 0.5% Triton X-100 for 15 min. After blocking with primary antibody dilution buffer for 1 h at room temperature, a mouse anti-α-actinin primary antibody (1:1000; Cat. ab9465, Abcam, USA) was applied overnight at 4° C. After two washes in PBS, the cells were incubated with an Alexa Fluor 555 conjugate secondary antibody (1:1000; Cat. 4409, CST, USA) for 2 h at room temperature. Cell nuclei were stained with DAPI for 5 min at room temperature and fluorescence images collected on an inverted microscope (BX63, Olympus) at 630x magnification.

Dual-luciferase reporter assay

HEK 293T cells were cultured in 24-well plates and co-transfected at ~70% confluence with miR 224-5p mimics and PGL3 luciferase plasmids containing WT, negative control (NC), or mutated TXNIP 3’UTR sequences. After 12 h, the cells were re-cultured in 96-well luciferase assay plates. The ratio of firefly to Renilla luciferase activity was detected 36 h later using a Dual-GLO^TM Luciferase Assay System (Cat. E2920, Promega, USA).

Real-time PCR

Total EV-associated RNA was extracted with RNAiso Plus extraction reagent following the manufacturer's instructions (Cat. 9108, Takara, Dalian, China). Stem-loop primers (purchased from Ribobio Biotech) were used to produce cDNA from miRNA using a PrimeScript RT reagent Kit (Cat. RR047A, Takara). The miRNA cDNA was amplified using TB Green® Premix Ex Taq™ II (Cat. RR820L, Takara) on an ABI-7500 Real-Time PCR Detection System (Applied Biosystems, USA). U6 snRNA was used as internal control.

Western blotting

Proteins were extracted from cardiac tissues and cardiomyocytes using Radio Immunoprecipitation Assay (RIPA). Ten mg of total protein was separated by 12% SDS-PAGE at 80 V for 1.5 h and then transferred to a PVDF membrane (Cat. IPVH00010, Millipore, USA) applying 300 mA for 1 h. After blocking, membranes were incubated with primary antibodies at 4° C overnight, washed, and treated with suitable secondary antibodies at room temperature for 1 h. Primary antibodies included rabbit anti-TSG101 (1:2000; Cat. ab125011, Abcam), rabbit anti-CD9 (1:2000; Cat. ab92726, Abcam), rabbit anti-TXNIP (1:1000; Cat. ab188865, Abcam), rabbit anti-ASC (1:1000; Cat. 67824, CST), rabbit anti-cleaved caspase-1 (1:1000; Cat. 89332, CST), rabbit anti-caspase-1 (1:1000; Cat. 24232, CST), rabbit anti-GATA4 (1:1000; Cat. ab134057, Abcam), rabbit anti-B-cell lymphoma-2 (Bcl-2; 1:1000; Cat. 3498, CST), rabbit anti-BCL2-associated X protein (BAX; 1:1000; Cat. 2772, CST), rabbit anti-caspase-3 (1:1000; Cat. 14220, CST), rabbit anti-α-actinin (1:1000; ab9465, Abcam), and rabbit anti-α-tubulin (1:1000; Cat. 2125, CST). Immunoreactive bands were visualized using an ECL substrate kit (Cat. ab65623, Abcam) and a two-color infrared fluorescence imaging system (Odyssey CLX, LICOR, USA). An anti-tubulin-α antibody was applied as internal control.

Statistical analysis

Data analysis was performed using SPSS 19.0 software. The Shapiro-Wilk test was applied to assess whether data conformed to normal distribution. Pearson’s chi-square test (n ≥ 5) or Fisher’s exact test (n < 5) with subsequent multiple comparisons using chi-square testing with Bonferroni correction were used for categorical variables. One-way ANOVA with subsequent post-hoc multiple comparisons test (Student-Newman-Keuls test) was applied for continuous variables. The Kruskal–Wallis test was applied for nonparametric testing of multiple independent samples and a Dunn-Bonferroni test used for post hoc comparisons.

Ethics approval

All animal procedures were approved by the Shanghai Ninth People’s Hospital institutional Ethics Committee (SH9H-2017-A39-1) and conducted in accordance with the guidelines of the Directive 2010/63/EU of the European Parliament.

Availability of data and material

The data that support the findings of this study are available from the corresponding author upon reasonable request.