The work has been reported in line with the ARRIVE guidelines 2.0.

Isolation of EPCs

Isolation of EPCs from bone marrow of Sprague-Dawley (SD) rats was performed as previously described [24] with minor modifications. In brief, young (3 − 6 weeks old) and old (64 − 68 weeks old) rats were anaesthetized via intraperitoneal injection of ketamine (80 mg/kg), and then sacrificed by cervical dislocation. The femurs and tibias were removed from the rats, and bone marrow was flushed out of the bones using PBS supplemented with 5 mM ethylene diamine tetraacetic acid (EDTA). The mononuclear cells in bone marrow were isolated using density-gradient centrifugation with Percoll solution (GE Healthcare, Leics, UK), and then incubated with Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 15% fetal bovine serum (FBS; Invitrogen, Grand Island, NY, USA), 100 U/mL penicillin and 100 µg/mL streptomycin for 24 h. The non-adherent cells were collected and seeded into gelatin-coated dishes. Upon being grown to 80% confluence, the cells were harvested by digestion with 0.25% trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA, USA). The cells were then incubated with mouse anti-rat CD34 and rabbit anti-rat VEGFR-2 antibodies (1:100; Santa Cruz Biotech, Dallas, TX, USA) at 4 oC for 50 min, followed by incubation with Alexa Fluor 647-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Jackson, West Grove, PA, USA) at 4 oC for 30 min. CD34+VEGFR-2+ cells in the mononuclear cells were sorted using a Beckman MoFlo™ XDP FACS (fluorescence-activated cell sorter; Beckman Coulter, Fullerton, CA, USA). To assess the differentiation potential of EPCs, the sorted cells were seeded in gelatin-coated dishes and incubated in the medium supplemented with 10 ng/mL VEGF (Peprotech, Rocky Hill, NJ, USA) and 15% FBS. Differentiation of the cells into the endothelial cells was determined by detecting of CD31 expression through immunostaining after 2 weeks of induction.

Detection of ALK-4 (activin-like kinase-4) expression

ALK-4, a member of Type-I TGF-β receptors, is expressed in EPCs [25]. ALK-4 expression in EPCs derived from young and old rats was detected using immunostaining and Western blotting respectively. For immunostaining, the cells were incubated with rabbit anti-rat ALK-4 antibody (1:250; Abcam, Cambridge, UK) at 4 oC overnight, followed by incubation with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200; Sigma-Aldrich, Saint Louis, MO, USA) at room temperature for 1 h. For Western blotting, the cells were lysed with RIPA buffer (Beyotime, Beijing, China). After quantifying with BCA protein assay kit (Beyotime), equal amounts of proteins were separated on 15% SDS-PAGE and then transferred onto PVDF membranes. The membranes were incubated with rabbit anti-rat ALK-4 antibody (1:1000; Abcam) and mouse anti-rat β-actin antibody (1:4000; Sigma-Aldrich) at 4 oC overnight, followed by incubation with HRP-linked goat anti-rabbit IgG (1:4000) and HRP-linked goat anti-mouse IgG (1:4000; Cell Signaling, Danvers, MA, USA) at room temperature for 1 h. Western blot bands were visualized by a Bio-Rad’s ChemiDoc System (BIO-RAD, Hercules, CA, USA). ALK-4/β-actin ratios were analyzed by ImageJ (National Institutes of Health, Bethesda, MD, USA) and normalized to β-actin. The experiment was repeated for three times.

Assessment of intracellular ROS (reactive oxygen species) generation

Cellular senescence can be induced with hydrogen peroxide (H2O2; Sinopharm, China) conditioning [26]. To determine the optimal concentration of H2O2 for inducing senescence without affecting the viability of the cells derived from young rats, the cells were treated with 100 µM, 250 µM and 500 µM H2O2 for 6 h respectively. Cell viability after H2O2 treatment was assessed using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan), and OD450 was measured by an Infinite M200 microplate reader (Tecan, Männedorf, Switzerland). 250 µM H2O2 was used for induction of senescence of the cells in the following experiments. The level of intracellular ROS was determined by ROS assay kit (Beyotime, Jiangsu, China). The cells were divided control, GDF11, H2O2 and H2O2 + GDF11 groups. In GDF11 group and H2O2 group, the cells were treated with 40 ng/mL GDF11 (Sigma-Aldrich) and 250 µM H2O2 respectively. In the H2O2 + GDF11 group, the cells were treated with both H2O2 (250 µM) and GDF11 (40 ng/mL). After treatment for 6 h, the cells were incubated with 10 µM peroxide-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime) at 37°C for 20 min. After permeating through cell membrane, DCFH-DA is hydrolyzed by intracellular esterases to DCFH. In the presence of ROS, DCFH is rapidly oxidized to fluorescent compound 2’,7’-dichlorofluorescein (DCF) [27]. DCF fluorescence was detected by microplate reader (Tecan). The experiment was repeated for three times.

SA-β-gal staining

SA-β-gal is a biomarker associated with the senescent phenotype. It catalyzes hydrolysis of artificial substrate X-gal, which produces a blue color in senescent cells [28]. The cells were divided into control, H2O2, H2O2 + GDF11 and H2O2 + GDF11 + SB-431542 groups. SB-431542 (MCE, Monmouth Junction, NJ, USA) is a small molecule and selective inhibitor to ALK4 and ALK5 [29]. At 6 h after incubation, the supernate was replaced with complete medium. In H2O2 + GDF11 and H2O2 + GDF11 + SB-431542 groups, GDF11 and SB-431542 (10 µM) were added, and the cells continued to be incubated for 48 h. Expression of SA-β-gal in the cells was examined with a SA-β-gal staining kit (Genmed Scientifics, Shanghai, China). The cells were fixed in 4% (v/v) formaldehyde for 8 min and then incubated with SA-β-gal staining solution at pH 6.0 for 12 h. The positive cells were counted using a phase contrast microscope. Three fields (20 ×) were randomly selected in each experiment. The experiment was repeated for three times.

Sudan black B staining

Sudan black B staining is used for examining lipofuscin accumulation in senescent cells [30]. The cells were divided into control, H2O2 and H2O2 + GDF11 groups. After treatment for 6 h, the cells were immersed in 70% ethanol for 2 min, and incubated with freshly prepared Sudan black B solution (0.7% in 70% ethanol; Sangon Biotech, Shanghai, China) for 8 min. Subsequently, the cells were immersed in 70% ethanol for a few times and counterstained with 0.1% nuclear fast red (Servicebio, Wuhan, China) for 10 min. Distribution of lipofuscin in the cells was viewed using a microscope. The experiment was repeated for three times.

Measurement of lipofuscin content

Intracellular lipofuscin content was measured as the autofluorescence intensity of the cells with flow cytometry as described previously [31]. Grouping of the cells is same as above described. 1 × 104 EPCs were harvested from each group and then washed twice in PBS by centrifugation. After the cells were resuspended in serum-free medium, the autofluorescence of the intracellular lipofuscin was analyzed using a flow cytometer (Beckman Coulter) with excitation wavelength of 488 nm and emission wavelength of 661 nm. The experiment was repeated for four times.

Detection of LC3 (microtubule-associated protein 1 light chain 3) expression

LC3 is mainly expressed on autophagosome and is a specific marker for autophagic structures [32]. For assessing LC3 expression on the autophagic structures, the cells were incubated with rabbit anti-rat LC3 antibody (1:200; Novus Biologicals, Littleton, CO, USA) at 4 oC overnight. Then, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Jackson) for 1 h at room temperature. LC3-positive puncta were viewed with a confocal laser scanning microscope (Leica, Mannheim, Germany) and counted in five randomly selected areas. The expression of LC3-I and LC3-II in the cells was examined by Western blotting. Briefly, proteins of the lysed cells were separated with 15% SDS-polyacrylamide gel and electrophoretically transferred to a PVDF membrane. The membrane was incubated with rabbit anti-rat LC3 antibody (1:500; Novus Biologicals) and mouse anti-rat β-actin antibody (1:4000; Sigma-Aldrich) at 4 oC overnight, followed by incubation with HRP-linked goat anti-rabbit IgG and HRP-linked goat anti-mouse IgG (1:4000; Cell Signaling) at room temperature for 1 h. The ratio of LC3-II/β-actin was analyzed using ImageJ (National Institutes of Health). The experiment was repeated for three times.

Transmission electron microscopy

The cells were divided into H2O2 and H2O2 + GDF11 groups. After being washed with PBS, the cells were fixed with 2.5% glutaraldehyde at 4 oC for 2 h, and then fixed with 1% osmium tetroxide. Subsequently, a series of dehydration was performed and ended with rinsing in 100% acetone. Then, the samples were embedded in epoxy resin, followed by being solidified in 37 oC, 45 oC and 60 oC for 12 h, 12 h and 24 h respectively. Ultrathin sections were prepared and then stained with 3% uranyl acetate and lead citrate. The autophagic structures in the cells were viewed using a CM120 transmission electron microscope (TEM; Philips, Eindhoven, Netherlands). The ratio of the cross-section area of the autophagic structures to that of the cytoplasm was calculated. The autophagic structures were examined in 200 cells for each group.

Atomic force and scanning electron microscopies

The SAP (AcN-RADARADARADARADARGDS-CONH2) was synthesized by Top-peptide Biotechnology (Shanghai, China). RGDS was designed as a cell adhesion motif. The SAP powder was dissolved in distilled water at 10 mg/mL. Then, the solution was diluted to a working concentration of 0.1 mg/mL and sonicated for 30 min. The sample were dropped on a mica, and left for 5 s at room temperature. Subsequently, the mica was gently rinsed twice with distilled water. SAP sample was air-dried at room temperature for 30 min. The nanofibers assembled by SAP were viewed using an atomic force microscope (AFM; Dimension Icon, Bruker, USA). In scanning electron microscopy, 10 µL SAP-PBS mixtures were coated on a cover slide and incubated at 37 oC for 30 min to allow the SAP to assembly into nanofibrous scaffolds. The cells were seeded on the SAP hydrogel and incubated at 37 oC for 2 h. The hydrogel containing the cells was treated as follows: fixed in 5% glutaraldehyde, dehydrated by gradual ethanol, dried in vacuum and coated with platinum. The SAP scaffolds and cells were viewed using a scanning electron microscope (SEM; Su8010, Hitachi, Tokyo, Japan).

Assessment of cytoprotective effect of SAP

EPCs isolated from young rats were seeded on the SAP hydrogel coated in 96-well plate, and incubated with DMEM supplemented 2% FBS in a sealed anoxia chamber (containing 1% O2, 5% CO2 and 94% N2) for 2 h. Viability of the cells was detected with CCK-8. The apoptotic cells and necrotic cells were stained with ethidium bromide and acridine orange (EB/AO), and counted using a fluorescence microscope. Concentration of VEGF in the supernate was detected with an enzyme-linked immunosorbent assay (ELISA) kit (Lichen, Shanghai, China). The experiments were repeated for six times.

Examination of the sustained release of GDF11 from SAP

10 µL GDF11 solution (10 µg/mL) was mixed with 10 µL SAP solution. Then, the mixed solution was added into 96-well plate and incubated at 37 oC for 30 min to allow the SAP hydrogel to encapsule GDF11. Subsequently, 200 µL PBS was added on SAP hydrogel. At 1 d, 3 d, 7 d, 14 d, 21 d and 28 d after incubation, the supernatants were drawn from the samples and replaced with PBS respectively. The concentration of GDF11 in the supernatant was measured with ELISA. The cumulative profile of GDF11 release was plotted. The experiments were repeated for six times.

Abdominal pouch assay

The abdominal pouch mimics the ischaemic and inflammatory tissue, and is convenient for examining oxidative stress-induced senescence of the implanted EPCs. The abdominal pouches of six SD rats (male, 200–250 g) was performed as previously described [33]. Briefly, after the rats were anesthetized with ketamine (80 mg/kg) and xylazine (5–10 mg/kg), the skin of the anterior abdominal wall at the median line was dissected, then the superficial fascia at both sides was bluntly dissected with a forceps to create two pouches. The blood vessels of the pouches were ligated carefully. The rats were divided into SAP and SAP + GDF11 groups. In SAP group, the cells derived from young rats were suspended with the SAP solution. In SAP + GDF11 group, 40 ng/mL GDF11 was mixed with SAP solution. Then, the cells were suspended with the mixed solution. The cells were seeded on poriferous polyethylene terephthalate membranes removed from the transwells (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated for 12 h. The cell-loaded membranes were implanted into the pouches, the cell side of the membranes was towards abdominal muscles. At 24 h after implantation, the membranes were harvested gently, and the senescent cells were examined with SA-β-gal staining and counted using a phase contrast microscope. Three fields (20 ×) were selected randomly in each membrane of each experiment. The experiment was repeated for three times.

Establishment of MI models and cell transplantation

Rat MI models were established as described previously [34]. Briefly, 44 female SD rats (200–250 g) were anesthetized with ketamine (80 mg/kg) and xylazine (5–10 mg/kg). After endotracheal intubation and ventilation using a rodent ventilator (Harvard Apparatus, Holliston, MA, USA), the heart was exposed through a 2-cm left lateral thoracotomy. The left anterior descending coronary artery (LADCA) was ligated. Successful infarction was determined by observing a pale discoloration in the anterior wall of the left ventricle (LV) and an obvious elevation of ST segment on electrocardiograms. Two rats died during and after LADCA ligation respectively. At 1 week after operation, 42 survived rats were randomly divided into 7 groups: sham, control, SAP, SAP + GDF11, EPCs, SAP + EPCs, SAP + GDF11 + EPCs. 10 µL SAP, 0.1 µg/mL GDF11 and 1 × 106 EPCs derived from young rats were used. Except for sham and control groups, the solutions in the other groups were diluted with PBS to 80 µL. The solutions were injected at the border of the infarcted myocardium through a 30-guage needle at 4 points (20 µL per point). In sham and control groups, the same volume of PBS was injected. For tracing the engrafted cells, the cells were transfected with green fluorescence protein (GFP)-lentivirus (Sangon Biotech, Shanghai, China) at a MOI of 30 for 72 h before transplantation. For maintaining body temperature, the rat was lain on an electric blanket in supine position during surgery.

Echocardiography

To evaluate the cardiac function, echocardiographic examination was performed before MI, and at 1 week after MI and 4 weeks post-transplantation. Echocardiograms of the rats were recorded by an ultrasonocardiograph (Visual Sonics, Toronto, Canada). The M-mode cursor was positioned to the parasternal line at the level of the papillary muscles. LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) were measured at least in 3 consecutive cardiac cycles. To evaluate LV systolic function, the ejection fraction (EF) and the fractional shortening (FS) were calculated as following formulae: EF (%) = (LVEDV – LVESV)/LVEDV × 100 and FS (%) = (LVEDD – LVESD)/LVEDD × 100. Two echocardiographers, blinded to the experimental treatment, acquired the images.

Histological examination

At 4 weeks after transplantation, the hearts were harvested and fixed with 4% paraformaldehyde overnight. After gradually dehydrated with 15% and 30% sucrose, the hearts were embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA, USA). Frozen sections were obtained from upper, middle and lower part ofthe hearts, and stained with Masson’s trichrome. The myocardial tissue was stained red, while the scar tissue was stained blue. The scar area was calculated as percentage of the infarct area in LV wall with ImageJ (Wayne Rasband, NIH, USA). The wall thickness of the infarct region was measured at the thinnest part of the infarcted LV.

The microvessels were examined by CD31 immunostaining. Densities of the microvessels in infarct and peri-infarct regions were measured by counting CD31-positive structures. At least 3 independent sections and 5 fields (20 x) on one section were selected randomly. To assess myocardial regeneration after transplantation, the infarct region was examined by cTnT and Cx43 immunostaining. The antibodies used for immunostaining were rabbit anti-GFP antibody (1:100; Santa Cruz Biotech), mouse anti-rat CD31 antibody (1:200; Abcam, Cambridge, MA, USA), mouse anti-rat cTnT antibody (1:200; Santa Cruz Biotech), rabbit anti-rat Cx43 antibody (1:100; Abcam), Alexa Fluor 594-conjugated goat anti-mouse IgG (1:400) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:400; Jackson). The antibodies used for immunostaining and Western blotting were listed in Table S1.

Senescence of the cardiomyocytes at peri-infarct region was assessed using SA-β-Gal staining and Sudan black B staining. Following SA-β-Gal staining, the sections were immersed in 70% ethanol for 1 min, and incubated in Sudan black B solution for 30 min at room temperature. Subsequently, the sections were immersed in 70% ethanol for a few times and counterstained with 0.1% nuclear fast red. SA-β-Gal-positive cardiomyocytes and lipofuscin granules were examined using a microscope.

The ultrastructures in the cardiomyocytes at peri-infarct region were examined using a CM120 TEM (Philips).

Identification of the senescent cells in the engrafted EPCs

Senescence of EPCs in the peri-infarct region at 4 weeks after transplantation was determined by combination of GFP immunostaining and SA-β-gal staining. Briefly, the frozen sections of the myocardium were treated with UV irradiation for 15 min, and then SA-β-gal staining of the sections was performed with SA-β-gal staining kit (Genmed Scientifics). GFP immunostaining was performed as above. The GFP+SA-β-gal+ cells in the survived cells (GFP+ cells) were counted using a fluorescence microscope. The experiment was repeated for six times.

Statistical analysis

Statistical analyses were performed with SPSS Statistics 23.0 (SPSS, Chicago, IL, USA), and graphical representations were generated using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). All data were presented as mean values ± standard deviation. Statistical differences between groups were evaluated using Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey post-hoc test. A P value of < 0.05 was considered statistically significant.