Cell culture
BM-MSCs and iPSC-MSCs were routinely cultured as described previously [23]. Briefly, MSCs were cultured in DMEM containing 10% fetal calf serum (FBS, GIBCO, 10270-106), 10 ng/mL epidermal growth factor (PeProTech, AF-100-15) and 5 ng/mL basic fibroblast growth factor (PeProTech, 100-18B). BM-MSCs at passage 3 ~ 4 and iPSC-MSCs at passage 6 ~ 8 were used in this study. Neonatal mouse cardiomyocytes (NMCMs) were isolated from heart tissue of neonatal mice (0- to 1-day-old) as described previously [24], then cultured at 37 °C in Claycomb Medium (Sigma, 51800) supplemented with 10% FBS.
Isolation and identification of MSC-EXOs
BM-MSC-EXOs and iPSC-MSC-EXOs were isolated from BM-MSCs and iPSC-MSCs and characterized as reported previously [15]. Briefly, 1 × 106 BM-MSCs or iPSC-MSCs were cultured in a 10-cm culture dish for 24 h and the culture medium then replaced with DMEM containing 10% exosome-depleted FBS (Systems Biosciences, EXO-FBS-250A-1). After a further 48 h culture, the supernatant was harvested and EXOs isolated using anion exchange chromatography. Next, MSC-EXOs were suspended in PBS and their concentration measured with a BCA assay kit (Thermo, 231227). To knockdown the level miR-9-5p in iPSC-MSC-EXOs, iPSC-MSCs were transfected with 50 nM miR-9-5p inhibitor and miR-9-5pKD-iPSC-MSC-EXOs isolated. The size and distribution of MSC-EXOs was assessed by Nanoparticle tracking analysis (NTA). The morphology of MSC-EXOs was determined by transmission electron microscopy (TEM) and exosomal surface markers determined by Western blotting.
Internalization of MSC-EXOs
To examine the uptake of MSC-EXOs by cardiomyocytes, MSC-EXOs were labeled with Dil (Beyotime, C1036) and then co-cultured with NMCMs for 24 h. After washing twice with PBS, NMCMs were fixed in 4% paraformaldehyde for 15 min and then stained with DAPI (Beyotime, C1005) for 15 min. Finally, NMCMs with Dil-labeled-MSC-EXOs were photographed under a confocal microscope.
SA-β-gal (senescence-associated β-galactosidase) assay
NMCM senescence was assessed by SA-β-gal staining according to the manufacturer’s instructions (Beyotime, #C0602). Briefly, NMCMs were cultured in a 6-well culture plate and treated with PBS, 10 μg/mL BM-MSC-EXOs or 10 μg/mL iPSC-MSC-EXOs under 1 μM DOX (MCE, HY-15142) challenge for 72 h. Next, cells were stained overnight with SA-β-gal solution at 37 °C without CO2. Subsequently, SA-β-gal positive NMCMs, evidenced by blue color were photographed under a microscope from five different fields of view. The percentage of senescent NMCMs was determined as the ratio of SA-β-gal positive NMCMs to total number of NMCMs.
MitoTracker staining
To detect the morphology of mitochondria in NMCMs, MitoTracker staining was performed. Briefly, NMCMs were seeded on 24-well plates with cover slides and the different treatments described above administered. Next, NMCMs were washed with PBS and incubated for 20 min at room temperature with DMEM containing 20 nM MitoTracker Green FM (Invitrogen, M7514). Subsequently, after washing with PBS, the stained NMCMs were randomly imaged from six fields and at least 300 NMCMs were counted in each group. Finally, the ratio of NMCMs with fragmented mitochondria to total number of NMCMs was calculated.
Transfection of miR-9-5p mimic or inhibitor
miR-9-5p mimic, miR-9-5p inhibitor and miR-Control were purchased from GenePharma Co., Ltd (Shanghai, China). Briefly, 1 × 106 iPSC-MSCs were seeded on a 10-cm culture dish and cultured for 24 h. Next, MSCs were transfected with 50 nM miR-9-5p mimic, inhibitor or miR-Control using Lipofectamine 2000 transfection reagent (Invitrogen, 11668027) and cultured for 48 h at 37 °C in a 5% CO2 incubator. Finally, the transfection efficiency was evaluated by qRT-PCR. The supernatant was harvested to collect EXOs according to protocols described above.
Luciferase assay
The 3′-UTR of human VPO1 (Vascular peroxidase 1) containing the miR-9-5p target site or the mutation in the seed region of the miR-9-5p binding site was inserted into the pGL3 luciferase reporter vector (Promega, Madison, WI, USA). 293 T cells were co-transfected with the reporter plasmid (pGL3-VPO1-3′-UTR or mutant VPO1-3′-UTR vector) and miR-9-5p mimics or inhibitors or miR-Control by Lipofectamine 2000 (Invitrogen, 11668027). Finally, luciferase activity was examined at 48 h after transfection using a Dual-Luciferase Reporter Assay System Kit (E1910, Promega).
Quantitative real-time PCR
Total RNA from NMCMs with or without different treatments, BM-MSC-EXOs and iPSC-MSC-EXOs was extracted with TRIzol reagent (Takara, 2270A). Reverse transcription was carried out using a PrimeScript RT Reagent Kit (Takara, RR037A). RT-PCR for miRNAs or VPO1 was determined using a One-Step TB Green® PrimeScript™ RT-PCR Kit according to the protocol (Takara, RR820A). GAPDH and U6 served as the internal reference. Relative expression of miRNAs and VPO1 mRNA was normalized and calculated by the 2 − ΔΔCt method.
Exosomal miRNA sequencing
Total RNA from BM-MSC-EXOs and iPSC-MSC-EXOs was extracted using a miRNeasy® Mini kit (Qiagen, 217004). The miRNA was sequenced using Illumina HiSeqTM 2500 (Genedenovo Co. Ltd, Guangzhou, China) as reported previously [15]. Raw reads were normalized and the expression of miRNAs analyzed to detect significant differences between BM-MSC-EXO and iPSC-MSC-EXO data sets. Differentially expressed miRNAs were identified through fold change > 1.5 and Q value < 0.001 with the threshold set for up- and down-regulated genes. Heat maps of differentially expressed miRNAs were generated by the omicshare cloud platform.
Western blotting
Total protein of NMCMs with different treatments and mouse heart tissue from different groups was extracted using a total protein extraction kit following the protocol (Bestbio, BB-3101) and protein concentrations measured with a BCA assay kit (Thermo, 231227). 30 μg protein from different groups was resolved on SDS-PAGE gel and then transferred to PVDF membranes. After blocking in TBST with 5% fat-free milk, the PVDF membranes were incubated overnight at 4 °C with the following primary antibodies: anti-TSG101 (Abcam, ab125011), anti-Alix (Abcam, ab186429), anti-p-Drp1 (Ser616) (CST, 3455), anti-Drp1 (CST, 14647), anti-p21 (Abcam, ab109199), anti-p53 (Abcam, ab26), anti-Mfn1 (Abcam, ab57602), anti-Mfn2 (Abcam, ab124773), anti-VPO1 (FineTest, FNab10858), anti-p-ERK (CST, 9101), anti-ERK (CST, 4695), and anti-GAPDH (CST, 2118). Membranes were then washed three times with TBST and incubated at room temperature with secondary antibodies for 1 h. Finally, the membranes were exposed in a dark room and the density of protein bands quantified by Image J software (National Institutes of Health, Bethesda, MD, USA).
Animal study
All animal procedures were approved by the Animal Research Committee of Guangdong Provincial People’s Hospital (No.KY-Z-2022-053-02). A mouse model of DIC was established in ICR mice (6 ~ 8 weeks) by intraperitoneal injection of DOX (3 mg/kg each time, six times over two weeks with a total cumulative dose = 18 mg/kg) as described previously [24]. In the control group, mice were intraperitoneally injected with an equal volume of PBS. Three doses of BM-MSC-EXOs, iPSC-MSC-EXOs or miR-9-5pKD-iPSC-MSC-EXOs (30 μg), suspended in 100μL PBS, were injected through the tail vein of DIC mice on days 9, 11, and 13, respectively. Cardiac function was measured by transthoracic echocardiography (Ultramark 9; Soma Technology, Bloomfield, CT, United States) on days 0, 7, 14 and 35. The mice were anesthetized using 2% isoflurane and chest hair removed. Next, all mice were placed on a heating pad (37 °C). The mouse heart was imaged using M-mode via a two-dimensional parasternal long axis with heart rate ranging from 350–500 beats/min. Left ventricle fractional shortening (LVFS) and ejection fraction (LVEF) were calculated. To study the cardioprotective effect of miR-9-5p on DIC, another DIC model was established and three doses of miR-9-5p agomir (30 mg/kg) or the same dosage of control agomir were injected through the tail vein of DIC mice on days 9, 11, and 13. Cardiac function was measured by transthoracic echocardiography on day 0 and 35.
Hematoxylin and eosin (H&E) staining
After heart function measurement on day 35, all mice were killed and heart tissue collected. Tissue was fixed, embedded, and cut into 5-μm sections. H&E staining was performed according to the manufacturer’s protocol (Servicebio, G1076). The percentage of cardiomyocyte vacuolization was calculated.
Sirius red staining
After echocardiographic measurement on day 35, Sirius red staining was performed according to the protocol. Images from 6 mice for each group were captured. The percentage fibrotic area was determined as the total fibrotic area/the total LV area × 100%.
TEM assay
The mitochondrial morphology in mouse heart tissue from different groups was examined by TEM assay as reported previously [15]. Images from 6 mice in each group were captured and at least 1000 mitochondria counted. Mitochondrial size was calculated using Image-Pro Plus software. Size < 0.6 μm2 was considered to indicate mitochondrial fragmentation.
Statistical analysis
Data are expressed as mean ± SD. Statistical analyses were performed by GraphPad Prism 9.3.0. Comparison between two groups was assessed using unpaired Student’s t-test, and comparison among more than two groups by one-way-ANOVA followed by the Bonferroni test. A p value < 0.05 was considered statistically significant.