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. 2021 Feb 2;10(5):756–772. doi: 10.1002/sctm.20-0456

Neurotrophin‐3 contributes to benefits of human embryonic stem cell‐derived cardiovascular progenitor cells against reperfused myocardial infarction

Wei Bi 1, Jinxi Wang 1, Yun Jiang 1, Qiang Li 1, Shihui Wang 1, Meilan Liu 1, Qiao Liu 1, Fang Li 1, Christian Paul 2, Yigang Wang 2, Huang‐Tian Yang 1,3,4,✉
PMCID: PMC8046156  PMID: 33529481

Abstract

Acute myocardial infarction (MI) resulting from coronary ischemia is a major cause of disability and death worldwide. Transplantation of human embryonic stem cell (hESC)‐derived cardiovascular progenitor cells (hCVPCs) promotes the healing of infarcted hearts by secreted factors. However, the hCVPC‐secreted proteins contributing to cardiac repair remain largely unidentified. In this study, we investigated protective effects of neurotrophin (NT)‐3 secreted from hCVPCs in hearts against myocardial ischemia/reperfusion (I/R) injury and explored the underlying mechanisms to determine the potential of using hCVPC products as a new therapeutic strategy. The implantation of hCVPCs into infarcted myocardium at the beginning of reperfusion following 1 hour of ischemia improved cardiac function and scar formation of mouse hearts. These beneficial effects were concomitant with reduced cardiomyocyte death and increased angiogenesis. Moreover, hCVPCs secreted a rich abundance of NT‐3. The cardioreparative effect of hCVPCs in the I/R hearts was mimicked by human recombinant NT‐3 (hNT‐3) but canceled by NT‐3 neutralizing antibody (NT‐3‐Ab). Furthermore, endogenous NT‐3 was detected in mouse adult cardiomyocytes and its level was enhanced in I/R hearts. Adenovirus‐mediated NT‐3 knockdown exacerbated myocardial I/R injury. Mechanistically, hNT‐3 and endogenous NT‐3 inhibited I/R‐induced cardiomyocyte apoptosis through activating the extracellular signal‐regulated kinase (ERK) and reducing the Bim level, resulting in the cardioreparative effects of infarcted hearts together with their effects in the improvement of angiogenesis. These results demonstrate for the first time that NT‐3 is a cardioprotective factor secreted by hCVPCs and exists in adult cardiomyocytes that reduces I/R‐induced cardiomyocyte apoptosis via the ERK‐Bim signaling pathway and promotes angiogenesis. As a cell product, NT‐3 may represent as a noncell approach for the treatment of myocardial I/R injury.

Keywords: apoptosis, cardiac repair, ERK‐Bim signaling pathway, human embryonic stem cell‐derived cardiovascular progenitor cells, neurotrophin‐3

Neurotrophin (NT)‐3 contributes to the cardioreparative effects of human embryonic stem cell (hESC)‐derived cardiovascular progenitor cells (hCVPCs) characterized by reduction of ischemia/reperfusion (I/R)‐induced cardiomyocyte apoptosis in adult mice via the extracellular signal‐regulated kinase (ERK)‐Bim signaling pathway and promotion of angiogenesis, resulting in the cardiac function improvement and fibrosis limitation in the infarcted hearts.

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Significance statement.

While implantation of human embryonic stem cell‐derived cardiovascular progenitor cells (hCVPCs) can promote the recovery of infarcted hearts via paracrine action, the contribution of secreted factors remains identified. The present study has revealed that neurotrophin (NT)‐3 is highly secreted by hCVPCs and the level is enhanced during mouse myocardial ischemia/reperfusion (I/R) injury. Injection of NT‐3 mimics the beneficial effect of hCVPCs in I/R hearts via suppressing cardiomyocyte apoptosis through modulation of the ERK‐Bim signaling pathway and promoting angiogenesis. These findings uncover a new function of NT‐3 in hCVPCs and I/R hearts and indicate a potential therapeutic role for cell products in the treatment of ischemic heart disease.

1. INTRODUCTION

Acute myocardial infarction (MI) resulting from coronary ischemia is a major cause of disability and death worldwide. Following MI, dead cardiomyocytes are replaced by fibrous scar tissue, causing ventricular remodeling and development of heart failure (HF). 1 , 2 To rescue ischemic myocardium and limit infarct size, timely and effective reperfusion in MI patients is the current therapeutic choice, however, this can cause additional damage, termed as ischemia/reperfusion (I/R) injury. 3 , 4 Therefore, identifying ways to protect cardiomyocytes against I/R injury during the early stage of reperfusion and clarifying the underlying protective mechanisms are necessary to promote the development of new therapeutic approaches for the treatment of ischemic heart disease.

Cell therapy by transplantation of stem/progenitor cells and their derived cardiovascular cells is a promising therapeutic approach for the treatment of ischemic heart disease. 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 Among the various transplantable cells, cardiac lineage cells derived from human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), show great potential in promoting healing of infarcted hearts. 5 , 7 , 13 , 14 , 15 , 16 , 17 Implantation of hESC‐derived cardiovascular progenitor cells (hCVPCs) during the subacute stage of I/R has been shown to improve cardiac function in rodent infarcted hearts 6 and increase engraftment in the infarcted myocardium of immunosuppressed nonhuman primates. 18 Moreover, no complications of arrhythmias, tumor formation, or immunosuppression‐related adverse events have been observed in the patients with advanced ischemic HF when SSEA1+ hCVPCs are transplanted. 5 Recently, we demonstrated that transplantation of SSEA1+ hCVPCs 19 , 20 at the acute‐phase of MI significantly reduces cardiomyocyte death, improves the recovery of left ventricular (LV) function, and/or limits the scar size in the murine infarcted hearts 15 and nonhuman primate MI models. 7 The beneficial effects of hCVPCs to the infarcted hearts are associated with paracrine factors, such as microRNAs 21 , 22 and long noncoding RNA MALAT1 23 in their secreted extracellular vesicles (EVs). Recently, we identified that hCVPC‐secreted cytokines, interleukin‐4 and interleukin‐13, modulate macrophage polarization toward a reparative phenotype in the infarcted hearts via activation of a signal transducer and activator of transcription 6. 15 However, it is unclear whether other proteins secreted from hCVPCs may exert synergistic cardioreparative effects, especially with respect to improving the cardiomyocyte survival during early reperfusion following myocardial ischemia.

Neurotrophins (NTs) are a family of highly conserved growth factors that serve a diverse set of functions in the nervous system 24 and cardiovascular cells. 25 , 26 NT‐3, a member of the neurotrophin family, 27 and its tropomyosin kinase receptor C (TrkC) are expressed in cardiovascular cells. 25 , 28 Genetic deficiency of Nt3 causes atrial and ventricular septal defects in mice. 29 In primary‐cultured neonatal rat cardiomyocytes (NRCMs), NT‐3 increases the mRNA level of cardiac hypertrophic genes and cell size, but it is downregulated in rat cardiac hypertrophy induced by pressure overload in vivo and in NRCMs stimulated by endothelin‐1. 28 In addition, adenovirus‐overexpression of NT‐3 stimulates the proliferation of capillary endothelial cells in the murine ischemic hind limbs via activation of protein kinase B (PKB/Akt) and endothelial nitric oxide synthase. 25 , 26 However, it is unknown whether NT‐3 is a paracrine factor of hCVPCs and little is known about the dynamic expression pattern and functions of NT‐3 in the adult heart with myocardial I/R injury.

The present study used a murine myocardial I/R model with injection of SSEA1+ hCVPCs into infarcted myocardium at the onset of reperfusion, combined with in vivo and in vitro manipulation of NT‐3 levels and extracellular signal‐regulated kinase (ERK) activation to investigate (i) the effects of hCVPCs on infarct healing when intramyocardially injected at the onset of reperfusion; (ii) the endogenous levels of NT‐3 protein in the adult murine hearts with and without myocardial I/R and its role in myocardial I/R injury; (iii) the signaling pathways mediated by NT‐3 in the protection of cardiomyocytes from I/R injury; and (iv) the contribution of NT‐3 to the beneficial effects of hCVPCs in promoting the healing of I/R hearts. Our findings have revealed a previously unrecognized paracrine factor of hCVPCs, its expression pattern and function in I/R hearts, the contribution to the cardioreparative effects of hCVPCs, and the underlying mechanisms. These findings not only provide new insights into the roles and mechanisms of the neurotrophin family member in tissue repair but also enrich our understanding of how hPSC‐derived cardiac lineage cells promote repair of the infarcted heart.

2. MATERIALS AND METHODS

2.1. Animals

Adult male C57BL/6 mice (The Shanghai Slac Laboratory Animal Co. Ltd, Shanghai, China) were used in the experiments. All procedures involving animals were performed in accordance with the Guidelines for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011) and approved by the Institutional Animal Care and Use Committee of Shanghai Institutes for Biological Sciences and Shanghai Institute of Nutrition and Health (Shanghai, China).

2.2. Induction of hCVPCs and preparation of hCVPC‐conditioned medium

The induction of hCVPCs was conducted following the protocol reported previously. 7 , 19 , 20 Detailed experimental methods are available in the Supplemental Materials section.

2.3. Flow cytometer analysis

Flow cytometer analysis was performed as described previously. 7 , 19 , 20 Detailed experimental methods are available in the Supplemental Materials section.

2.4. Immunocytochemical staining analysis

Cells were immunostained as described previously. 7 , 19 , 20 Detailed experimental methods are available in the Supplemental Materials section.

2.5. Myocardial I/R injury model and study designs

The myocardial I/R model was induced as previously described. 30 Briefly, male C57BL/6 mice at 10‐12 weeks‐old were anesthetized via intraperitoneal injection (i.p.) of 50 mg/kg sodium pentobarbital and mechanically ventilated using a volume regulated respirator (SAR830, Cwe Incorporated). Body temperature was maintained at 37°C throughout the surgical procedure. Ischemia was produced through ligation of the left anterior descending (LAD) coronary artery with an 8‐0 Prolene suture, while Sham group mice had a loose suture placed in the same position. After 1 hour of occlusion, the ligature was released and various treatments were performed at the onset of reperfusion in the following groups: (i) for effects of hCVPCs in I/R hearts, Sham group, I/R + Puramatrix (PM, reported to retain the transplanted cells or proteins in the injected site, 31 , 32 3‐D Matrix, Ltd) control group (10 μL 10% sucrose solution+10 μL 0.4% PM/heart), and I/R + hCVPC group (5 × 105 cells in 20 μL of 0.2% PM/heart). The PM and hCVPCs were injected into the peri‐infarct regions of I/R hearts at 3 sites; (ii) for functions of NT‐3 in I/R hearts, I/R + PBS (phosphate‐buffered saline, 20 μL/heart) group or I/R + hNT‐3 (human recombinant NT‐3) group (0.5 μg in 20 μL PBS/heart, GeneTex). PBS or hNT‐3 was injected into the peri‐infarct regions at 3 sites, followed by continuous release of PBS or hNT‐3 (0.5 μg d−1) for up to 1 week via the Micro‐Osmotic Pump (Model 1004, Alzet) implanted subcutaneously; (iii) for involvement of the ERK‐Bim signaling pathway in NT‐3‐regulated cardioprotection, I/R group, I/R + hNT‐3 group (0.5 μg in 20 μL/heart), I/R + SCH (ERK specific inhibitor SCH772984, Selleck) group, and I/R + hNT‐3 + SCH group. hNT‐3 was intramyocardially injected and SCH772984 (10 μg/20 μL/heart) was administrated (i.p.) at the onset of reperfusion; (iv) and for contributions of NT‐3 to the benefits of hCVPCs in I/R hearts, Sham group, I/R group (PM control), I/R + IgG (mouse immunoglobulin G, R&D) group, I/R + NT‐3‐Ab (NT‐3‐neutralizing antibody, R&D) group, I/R + hCVPCs group, I/R + hCVPCs+IgG group, and I/R + hCVPCs+NT‐3‐Ab group. IgG or NT‐3‐Ab at 300 ng/mL was intramyocardially injected together with hCVPCs (5 × 105 cells/heart) or PM into the peri‐infarct region at 3 sites (20 μL/heart). Hearts were harvest at day 1 or day 28 after I/R for analysis.

2.6. Echocardiography

Transthoracic echocardiography (Vevo 2100, Visual Sonics) with a 25 MHz imaging probe was used on to obtain serial ultrasound images in the anesthetized mice as previously described. 15 , 23 LV ejection fraction (LVEF), LV fractional shortening (LVFS), and LV systolic dimension (LVDs) were measured using the M‐mode at day 2, 7 and 28 after surgery. All measurements were averaged from three consecutive cardiac cycles in a blinded manner.

2.7. Construction of recombinant adenovirus

Recombinant adenoviruses (Ad) expressing short hairpin RNA were prepared as reported previously. 30 , 33 , 34 Detailed experimental methods are available in the Supplemental Materials section.

2.8. Adenovirus‐mediated gene delivery in vivo

Surgical procedures and adenoviral delivery were carried out as previously described. 30 , 34 Briefly, the surgery process was the same as described above and a 30‐gauge needle containing 30 μL of diluted the scramble (sh‐scramble/negative control, shNC) or the short hairpin RNA against mouse NT‐3 (shNT‐3) (3 × 1010 pfu/mL) was injected into the myocardium of LV at 3 sites. The efficiency of adenoviral downregulation of NT‐3 was assessed by Western blot at day 4 after adenovirus injection. The same mice were then subjected to myocardial I/R surgery.

2.9. Immunohistochemical staining

Immunohistochemical (IHC) analysis was performed as previously described. 15 , 23 Detailed experimental methods are available in the Supplemental Materials section.

2.10. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive (TUNEL+) cardiomyocytes in I/R hearts and isolated cells were evaluated using an In situ Cell Death Detection Kit (Roche Applied Science, Germany), co‐stained with anti‐cTnT antibody. The percentage of TUNEL+ cardiomyocytes was blindly quantified as the ratio of TUNEL+cTnT+ cardiomyocytes to the total cTnT+ cells as described previously. 15 , 23

2.11. Analysis of infarct size

Infarct size was measured by using 1% w/v of 2, 3, 5‐triphenyltetra‐zolium chloride (TTC, T8877, Sigma‐Aldrich) as previously described. 30 Detailed experimental methods are available in the Supplemental Materials section.

2.12. Masson's trichrome staining and FITC‐conjugated wheat germ agglutinin staining

Heart frozen sections were stained after 28 days of reperfusion with Masson's trichrome as previously described. 15 , 23 Briefly, morphometric parameters measured on five sections each heart, including total LV area and scar area, were blindly analyzed from ligated to apical position of the heart and the scar size was calculated as the total scar area divided by the LV area with Image J. The FITC‐labeled wheat germ agglutinin antibody (1:100, Sigma, L4895) was used to perform immunostaining on LV cross sections from the same level of various hearts. The images at each section were captured by Zeiss fluorescence microscope and 60‐90 myocytes from 2‐3 independent fields per section in each heart were quantified using Image J as reported previously. 14 , 35

2.13. Lactate dehydrogenase activity

Lactate dehydrogenase (LDH) activity was analyzed with an LDH Cytotoxicity Assay Kit (Beyotime Institute of Biotechnology) according to the supplier's instructions as previously described. 34 , 36 Detailed experimental methods are available in the Supplemental Materials section.

2.14. Isolation and culture of adult mouse cardiomyocytes and adult mouse cardiac fibroblasts

Adult mouse cardiomyocytes (AMCMs) were isolated from the hearts of adult male C57BL/6 mice and cultured as previously described. 30 Detailed experimental methods are available in the Supplemental Materials section.

2.15. Simulated I/R in isolated AMCMs

A cellular simulated I/R (sI/R) injury model was used for isolated AMCMs as previously reported. 37 , 38 Briefly, isolated AMCMs were incubated with ischemic buffer for 1 hour, followed by 1 hour of reperfusion using culture medium in 21% O2 and 5% CO2. The various treatments were started at the beginning of reperfusion lasting for 1 hour. Detailed experimental methods are available in the Supplemental Materials section.

2.16. Western blot protein array analysis

Total proteins from the infarct and border zones of LV tissues or AMCMs were extracted and analyzed as previously reported. 15 , 23 , 30 , 39 Detailed experimental methods are available in the Supplemental Materials section.

2.17. Quantitative reverse transcription polymerase chain reaction

Quantitative reverse transcription polymerase chain reaction (RT‐qPCR) was performed as described previously. 15 , 19 , 23 Detailed experimental method is available in the Supplemental Materials section.

2.18. Statistical analysis

Data are presented as mean ± SEM. Statistical significance was analyzed by using unpaired Student's t‐test for comparisons between two groups, one‐way analysis of variance (ANOVA) for comparisons between more than two groups when there was one independent variable), and two‐way ANOVA for the analysis of two independent variables or echocardiographic data. One‐way and two‐way ANOVA were followed with Bonferroni's multiple comparison. Statistically analysis was performed with Graphpad Prism software (version 6.1). A P‐value <.05 was considered statistically significant.

3. RESULTS

3.1. Therapeutic benefits of hCVPCs in mice with myocardial I/R injury

The H9 hESCs (WiCell)‐generated SSEA1+ hCVPCs displayed CVPC markers MESP1, ISL1, MEF2C, GATA4, and NKX2.5 (Figure S1) as previously reported. 7 , 15 , 19 , 20 , 23 Mice injected with hCVPCs in the peri‐infarct region at the onset of reperfusion following 1 hour of ischemia (Figure 1A) showed comparable LVEF, LVFS and LVDs with those of the I/R group at day 2 post‐I/R, whereas the hCVPC treatment significantly improved these parameters that were worsened in the I/R control group during the 4‐week follow‐up period (Figure 1B). Consistently, Masson's trichrome staining analysis at day 28 post‐I/R showed that scar size in the hCVPC‐treated hearts was significantly smaller than the control group (Figure 1C). As the reduction of scar size may partially result from a reduction in cell death and/or improvement of angiogenesis, we analyzed infarct size and apoptotic cardiomyocytes at day 1 post‐I/R. The I/R‐PM and hCVPC groups showed comparable area at risk (AAR) to LV, but the infarct area (Figure 1D), serum LDH activity (an index of cell death, Figure 1E), and TUNEL+ cardiomyocytes (Figure 1F) were significantly attenuated in the hCVPC group compared with those in the PM group. Moreover, the I/R‐increased cleaved caspase‐3 (cCas‐3) in the infarct and border zones was reduced in the hCVPC treated group (Figure 1G). IHC staining at day 28 post‐I/R showed an increased number of CD31+ endothelial cells (Figure S2A) and α‐smooth muscle actin positive (α‐SMA+) vessels (Figure S2B) in the infarct and border zones of hCVPC‐treated I/R hearts compared with the PM group.

FIGURE 1.

FIGURE 1

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hCVPC delivery improves cardiac function and cardiomyocyte survival in ischemia/reperfusion (I/R) hearts. A, Schematic of hCVPC transplantation and analysis. B, Left ventricle (LV) ejection fraction (LVEF), LV fractional shortening (LVFS) and LV systolic dimension (LVDs) measured by echocardiography (Echo). n = 10. C, Representative cross‐sectional images and quantitative data of heart scar area stained with Masson's trichrome at day 28 post‐I/R. n = 9. Scale bars = 1 mm. D, Representative and quantification of 2,3,5‐triphenyte‐trazoliumchloride (TTC)/Evans blue staining for infarct size in the hearts at day 1 post‐I/R. n = 4. Scale bar = 1 mm. E, Fold changes of lactate dehydrogenase (LDH) activities in PM (Puramatrix) and hCVPCs groups compared with the one in Sham mice. n = 4‐5. F, Representative and quantification of immunohistochemical (IHC) staining for terminal deoxynucleotidyl transferase dUTP nick end labeling positive (TUNEL+) cardiomyocytes in the infarct and border zones of hearts at day 1 post‐I/R. n = 4. Scale bar = 50 μm. G, Representative and averaged Western blot analysis for caspase‐3 (Cas‐3), cleaved Cas‐3 (cCas‐3) and GAPDH in the infarct and border zones of hearts at day 1 post‐I/R. n = 3. *P < .05, **P < .01, ***P < .001 vs the Sham group; # P < .05, ### P < .001 vs the I/R + PM group. ns, nonsignificant

3.2. NT‐3 is highly secreted from hCVPCs and promotes infarct healing of I/R hearts

To determine whether the beneficial effects of hCVPCs on cardiovascular cells in the I/R hearts are related to the paracrine action of hCVPCs, the hCVPC‐secretome was analyzed as previously described. 15 Among the secreted proteins of hCVPCs, an abundance of NT‐3 was detected in the hCVPCs and preparation of hCVPC‐conditioned medium (hCVPC‐CdM) (Figure S3A). This was confirmed by RT‐qPCR (Figure S3B) and ELISA analysis (Figure S3C). To determine the role of exogenous NT‐3 in the I/R hearts, hNT‐3 (0.5 μg/20 μL) was then injected into the peri‐infarct region of the I/R hearts at the beginning of reperfusion followed by continuous subcutaneous administration of NT‐3 (0.5 μg d−1) up to day 7 of I/R (Figure 2A). LVEF, LVFS, and LVDs were comparable between the PBS and hNT‐3‐injected groups without I/R insult (Figure S4) or at day 2 post‐I/R, whereas hNT‐3 treatment improved LVEF and LVFS in the I/R hearts during the 4‐week follow‐up period (Figure 2B). Concurrently, I/R hearts developed smaller infarct scars (Figure 2C) and less myocyte hypertrophy (Figure S5) in the hNT‐3‐treated group than those in the control group. Moreover, the AAR of LV at day 1 post‐reperfusion was comparable between the PBS and hNT‐3 treatment groups, whereas the infarct size (Figure 2D), LDH release (Figure 2E), the number of TUNEL+ cardiomyocytes in the infarct and border zones (Figure 2F), and cCas‐3 (Figure S6) were significantly reduced in the hNT‐3 group. Furthermore, the number of CD31+ endothelial cells (Figure 2G) and the density of SMA+ blood vessels in the infarct and border zones (Figure 2H) were higher in the hNT‐3‐treatment group than those in the PBS control group at day 28 post‐I/R.

FIGURE 2.

FIGURE 2

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Exogenous hNT‐3 promotes infarct healing of I/R hearts. A, Schematic of hNT‐3 treatment regime in C57BL/6 wild type (WT) I/R mice. B, LVEF, LVFs and LVDs measured by echocardiography. n = 9. C, Representative cross‐sectional images and quantitative data of scar area stained with Masson's trichrome at day 28 post‐I/R. n = 6. Scale bars = 1 mm. D, Representative and quantification of TTC/Evans blue staining for infarct size in the hearts at day 1 post‐I/R. n = 4‐5. Scale bar = 1 mm. E, Fold changes of LDH activities in LV serum between I/R + PBS and I/R + hNT‐3 group compared with Sham mice. n = 8. F, Representative and quantification of IHC staining for TUNEL+ cardiomyocytes in the infarct and border zones of hearts at day 1 post‐I/R. n = 3. Scale bar = 50 μm. G, IHC staining and quantitative data for CD31+ endothelial cells in the infarct and border zones at day 28 post‐I/R. n = 5. Scale bar = 50 μm. H, IHC staining and quantitative data for α‐smooth muscle actin positive(α‐SMA+) blood vessels in the infarct and border zones at day 28 post‐I/R. Scale bar = 50 μm. *P < .05; **P < .01; ***P < .001; ns, nonsignificant

3.3. Exogenous hNT‐3 protects the cardiomyocytes from sI/R injury

To determine whether the cardioprotective effects of hNT‐3 are related to its action in the cardiomyocytes, we induced sI/R injury in the AMCMs as reported previously. 37 , 38 Notably, the sI/R injury‐induced cardiomyocyte death (in round shape, Figure 3A) and LDH release (Figure 3B) at the 1 hour of reperfusion were decreased by hNT‐3 in a concentration‐dependent manner. Concomitantly, hNT‐3 at 10 ng/mL significant decreased the number of TUNEL+ AMCMs (Figure 3C) and the cCas‐3 level (Figure 3D) induced by sI/R injury.

FIGURE 3.

FIGURE 3

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Protective effects of exogenous hNT‐3 in the adult mice cardiomyocytes (AMCMs) subjected to 1 hour of simulated I/R (sI/R) injury. A, Representative images of AMCM morphology and quantitative percentages of round/total cells at normoxia or 1‐hour post‐sI/R. n = 5‐11. Scale bar = 100 μm. B, LDH activities in AMCM culture media. n = 5‐8. C, Representative and quantification of IHC staining for TUNEL+ AMCMs. n = 5. Scale bar = 50 μm. D, Representative and averaged Western blot analysis in AMCMs at 1‐hour post‐sI/R. n = 3. **P < .01; ***P < .001

3.4. NT‐3 is an endogenous protective factor in the heart against I/R injury

To determine whether NT‐3 exists in the adult murine hearts and contributes to the alleviation of myocardial I/R injury, NT‐3 was knocked down in the heart by intramyocardial injection of Ad‐shNT‐3 at 4 days before the myocardial I/R (Figure 4A). NT‐3 protein was detected in isolated AMCMs and adult mouse cardiac fibroblasts (AMCFs) (Figure S7A), whereas levels in the infarct and border zones of I/R hearts decreased at the beginning of reperfusion but significantly increased by day 3 and day 7, and returned to the baseline by day 28 post‐reperfusion (Figure S7B). Compared with the Ad‐shNC control group, the NT‐3 protein level in the LV was downregulated by 30% in the heart by intramyocardial injection of Ad‐shNT‐3 at 4 days before the myocardial I/R (Figure S8A), whereas the LVEF, LVFS, and LVDs were similar between the two groups at day 4 post‐infection before I/R surgery (Figure S8B). At day 2 post‐I/R, LVEF, LVFS, and LVDs did not differ between the two groups (P > .05), whereas lower LVEF and LVFS were detected in NT‐3 knockdown mice at day 28 post‐I/R when compared with those of the Ad‐shNC mice (Figure 4B). The NT‐3 knockdown group had large infarct scars compared with Ad‐shNC control mice at day 28 after I/R (Figure 4C). Moreover, larger infarct scars (Figure 4D), higher LDH release (Figure 4E), increased TUNEL+ cardiomyocytes (Figure 4F) and cCas‐3 (Figure S8C) in the infarct and border zones at day 1 of I/R were detected in the NT‐3 knockdown mice compared with those in the Ad‐shNC control groups, though they had comparable AAR to LV (Figure 4D). The Ad‐shNT‐3 group showed a lower number of CD31+ endothelial cells (Figure 4G) and lower density of α‐SMA+ blood vessels (Figure 4H) in the infarct and border zones of I/R hearts than those in the Ad‐shNC control group. These data support our hypothesis that NT‐3 is an endogenous protective factor in the heart against I/R injury.

FIGURE 4.

FIGURE 4

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NT‐3 is an endogenous protective factor in the heart against I/R injury. A, Schematic of treatment regime in I/R mice. B, LVEF, LVFS and LVDs measured by echocardiography. n = 9. C, Representative cross‐sectional images and quantitative data of scar area stained with Masson's trichrome at day 28 post‐I/R. n = 6‐7. Scale bars = 1 mm. D, Representative and quantification of TTC/Evans blue staining for the infarct size at day 1 post‐I/R. n = 4‐5. Scale bar = 1 mm. E, Fold changes of LDH activities in the LV serum at day 1 of I/R. n = 5‐6. F, Representative and quantification of IHC staining for TUNEL+ cardiomyocytes in border and infarct zones of hearts at day 1 post‐I/R. n = 3. Scale bar = 50 μm. G, IHC staining and quantitative data for CD31+ endothelial cells in the infarct and border zones at day 28 post‐I/R. n = 5. Scale bar = 50 μm. H, IHC staining and quantitative data for α‐SMA+ blood vessels in the infarct and border zones at day 28 post‐I/R. n = 5. Scale bar = 50 μm. Ad‐shNC, adenovirus‐short hairpin RNA of negative control; Ad‐sh‐NT‐3, adenovirus‐short hairpin RNA of mouse NT‐3. *P < .05; **P < .01 vs the Ad‐shNC group or as indicated in figures; ns, nonsignificant

3.5. NT‐3 protects cardiomyocytes from I/R‐induced apoptosis via activating ERK‐Bim signaling pathway

Then the levels of phosphokinases involved in the regulation of cell survival were compared between the PBS‐ and hNT‐3‐treated I/R hearts. Among the 43 screened phosphokinases, hNT‐3‐treatment enhanced the I/R‐increased phosphorylation of ERK (P‐ERK) in 1 day post‐I/R hearts (Figure S9A). The hNT‐3‐increased P‐ERK levels (Figure 5A) but not phosphorylation of Akt and P38 (Figure S9B) at 1 day post‐I/R, which was confirmed using Western blot analysis. Myocardial I/R‐enhanced protein level of Bim (a downstream target of P‐ERK and an activator of mitochondrial apoptosis 40 ) was significantly reduced by hNT‐3, whereas the phosphorylation of RSK (a P‐ERK downstream effector suppressing apoptosis 41 ) remained unchanged (Figure 5A). Activation of ERK by hNT‐3 was further confirmed in AMCMs without sI/R (Figure 5B) or suffering 30 minutes of reperfusion following 1 hour of simulated ischemia (Figure 5C). Moreover, the myocardial I/R‐enhanced P‐ERK was reversed to the level as seen in the sham group following NT‐3 knockdown in the I/R hearts (Figure 5D). To further confirm whether NT‐3‐mediated inhibition in cardiomyocyte apoptosis via activation of the ERK‐Bim signaling pathway, a ERK specific inhibitor SCH772984 was injected intraperitoneally in mice at the onset of reperfusion. SCH772984 inhibited the hNT‐3‐enhanced ERK phosphorylation level at 10 μg/20 μL (Figure S10). Concomitantly, the AAR to LV was comparable among various groups, whereas SCH772984 enlarged the infarct size compared with the control group and canceled the protective effect of hNT‐3 in the limitation of infarct size (Figure 6A). Moreover, I/R‐induced LDH activity in serum (Figure 6B), TUNEL+ cardiomyocytes in the infarct and border zones (Figure 6C,D), and cCas‐3 (Figure 6E) were increased following SCH772984 treatment; and the protective effects of NT‐3 on these parameters were reversed by SCH772984 at day 1 post‐reperfusion. hNT‐3 enhanced P‐ERK at day 1 post‐reperfusion was blocked by SCH772984 with the oppositely altered levels of Bim (Figure 6F).

FIGURE 5.

FIGURE 5

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NT‐3 activates phosphorylation of ERK in the I/R hearts and AMCMs. A, Representative and averaged Western blot analysis in the mice hearts at 1 day post‐I/R. n = 3. B, Representative and averaged Western blot analysis in the normoxic AMCMs with or without 10 ng/mL hNT‐3 treatment. n = 3. C, Representative and averaged Western blot analysis in the AMCMs subjected to sI/R injury with or without 10 ng/mL hNT‐3 treatments. n = 3. D, Representative and averaged Western blot in the NT‐3 knockdown hearts at day 1 post‐I/R. n = 3. *P < .05; **P < .01; ***P < .001; ns, nonsignificant

FIGURE 6.

FIGURE 6

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NT‐3 protects the heart against I/R‐induced cardiomyocyte apoptosis via the ERK‐Bim signaling pathway. A, Representative and quantification of TTC/Evans blue staining for infarct size in the hearts among I/R, I/R + hNT‐3 (0.5 μg), I/R + SCH (10 μg) and I/R + hNT‐3 + SCH groups at day 1 post‐I/R. n = 3‐6. Scale bar = 1 mm. B, Fold changes of LDH activities in the LV serum compared with the one in Sham mice hearts at day 1 post‐I/R. n = 4‐6. C and D, Representative and quantification of IHC staining for TUNEL+ cardiomyocytes in the infarct and border zones of hearts at day 1 post‐I/R. n = 4. Scale bar = 50 μm. E and F, Representative and averaged Western blot of various proteins in the infarct and border zones of day 1 post‐I/R hearts. n = 3. SCH, ERK inhibitor SCH772984. *P < .05; **P < .01; ***P < .001; ns, nonsignificant

The involvement of ERK‐Bim signaling pathway in the protection of NT‐3 was then confirmed on AMCMs subjected to sI/R injury. SCH772984 decreased sI/R‐ and hNT‐3‐enhanced P‐ERK levels in a concentration‐dependent manner (Figure S11A). Concomitantly, SCH772984 cancelled the hNT‐3‐afforded protection in the reduction of sI/R‐increased round cell proportion (Figure S11B), increased protein levels of Bim, and cCas‐3 (Figure S11C).

3.6. NT‐3 contributes to the reparative effects of hCVPCs in I/R hearts via activating the ERK‐Bim signaling pathway

Next, we determined whether NT‐3 contributes to the cardioreparative effect of hCVPCs by neutralization of NT‐3 with its antibody (Figure S12). sI/R‐induced increase of LDH activity AMCMs was significantly suppressed by hCVPC‐CdM, whereas this protection was cancelled by NT‐3‐Ab in a concentration‐dependent manner but not the IgG control (Figure S12). Furthermore, hCVPCs were injected together with the IgG or NT‐3‐Ab into the peri‐infarct region of I/R hearts. The ratios of AAR to LV were comparable among the various groups, whereas the hCVPC‐reduced infarct size at day 1 post‐I/R was reversed by the NT‐3‐Ab but not the IgG control (Figure 7A). Similar changes of serum LDH activity at day 1 reperfusion were observed (Figure 7B). Concomitantly, the hCVPC‐reduced TUNEL+ cardiomyocytes (Figure 7C,D) and cCas‐3 level (Figure 7E) in the infarct and border zones of I/R hearts were reversed to the level in the I/R group. Moreover, the NT‐3‐Ab treatment cancelled hCVPC‐mediated enhancement in the P‐ERK and suppression in the Bim level in the I/R hearts (Figure 7F).

FIGURE 7.

FIGURE 7

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NT‐3 contributes to the reparative effects of hCVPCs in the I/R hearts via activating the ERK‐Bim signaling pathway. A, Representative and quantification of TTC/Evans blue staining for infarct size in the hearts at day 1 post‐I/R. n = 3‐6. Scale bar = 1 mm. B, LDH activities in the LV serum at day 1 of I/R. n = 3‐4. C and D, Representative and quantification of IHC staining for TUNEL+ cardiomyocytes in the border zones of infarcted hearts at day 1 post‐I/R. n = 4. Scale bar = 50 μm. E, Representative and averaged Western blot analysis for Cas‐3, cCas‐3 and GAPDH in the infarct and border zones of day 1 post‐I/R hearts. n = 3. F, Representative images and averaged Western blot analysis for P‐ERK, T‐ERK, Bim, and GAPDH in the infarct and border zones of day 1 post‐I/R hearts. n = 3. *P < .05; **P < .01; ***P < .001; ns, nonsignificant

4. DISCUSSION

These results confirm that implantation of hCVPCs at the onset of reperfusion can improve cardiac function and reduce fibrosis in I/R hearts. These beneficial effects are at least related to the improvement of existing cardiomyocyte survival and angiogenesis in I/R hearts. Moreover, we found that hCVPCs highly secrete NT‐3 and delivery of hNT‐3 to I/R hearts or to sI/R AMCMs at the beginning of reperfusion mimics the beneficial effects of hCVPCs, whereas NT‐3 antibody cancels the beneficial effects of hCVPCs. Furthermore, we showed that NT‐3 exists in AMCMs and AMCFs, and downregulation of endogenous NT‐3 in murine hearts aggravates myocardial I/R injury, accompanied with enhanced cardiomyocyte apoptosis and reduced vessel density. Mechanistically, in addition to the enhancement of vessel density in the infarcted hearts, hCVPC‐secreted NT‐3 and endogenous NT‐3 attenuate I/R‐induced apoptosis of cardiomyocytes through the activation of ERK, thereby reducing the level of Bim, a key mitochondrial apoptosis activator, and promoting the healing of infarcted hearts. These findings reveal a previously unrecognized paracrine factor of hCVPCs and identify new roles of hCVPC‐secreted endogenous protein NT‐3 in cardiac protection against myocardial I/R injury. The findings also provide new insights into the cardioprotective mechanisms of NT‐3 in cardiac repair.

Several novel observations have been obtained from the present study. Firstly, we demonstrated that NT‐3 is abundantly secreted from hCVPCs and critically contributes to hCVPC‐mediated cardiac repair following I/R injury. NT‐3 is produced in neuronal cells, 24 cardiovascular cells, 25 and stem cells, such as human mesenchymal stem cells (MSCs), 42 neural stem cells, 43 and hESCs. 44 The generation of NT‐3 in multiple cells indicates that NT‐3 serves pivotal functions in the maintenance of normal development and organ function in both physiological and pathological conditions via autocrine and paracrine mechanisms. It has been shown that NT‐3 is an essential factor for mammalian great vessel and cardiac development, 25 , 29 promotes angiogenesis 26 and cardiomyocyte hypertrophy. 28 Our findings are the first to identify NT‐3 as an important paracrine factor in hCVPC‐mediated cardioreparative effects. This is supported by the high concentration of NT‐3 in hCVPC‐CdM and the abolishment of cardioreparative effects of hCVPCs in I/R hearts and the protection of hCVPC‐CdM in sI/R cardiomyocytes by NT‐3‐Ab. It has been reported that MSC transplantation‐mediated improvement of functional outcome in animal models of neurological disorders is partially associated with the secretion of NT‐3, 42 indicating that NT‐3 appears to contribute to the reparative effects associated with stem cell therapy. Therefore, to thoroughly dissect the secretome of hPSC‐derived cardiac lineage cells and to elucidate how these factors and EVs/exosomes protect I/R hearts would be of great value for the development of new therapeutic approaches for ischemic heart disease.

Secondly, results from this study reveal the dynamic expression pattern and the role of endogenous NT‐3 in I/R hearts. Although NT‐3 is detected in adult rat cardiomyocytes, 28 the dynamic pattern of NT‐3 protein levels during myocardial I/R is unknown. Our results demonstrated the existence of NT‐3 protein in AMCMs and AMCFs. The NT‐3 protein levels tend to decrease at the beginning of reperfusion and then upregulate in the infarct and border zones at day 3 to day 7 post‐reperfusion in adult murine hearts. Similarly, upregulation of NT‐3 mRNA is detected at day 3 following ischemia in murine skeletal muscle. 26 Moreover, the expression of nerve growth factor increases following hypoxia/reoxygenation in cultured rat neonatal cardiomyocytes. 45 These observations suggest that enhancement of NT‐3 represents an endogenous protective mechanism for repairing damaged tissue. This is supported by our data showing that downregulation of endogenous NT‐3 in adult murine hearts significantly deteriorates myocardial I/R injury, characterized by worsened LV function and enhanced fibrosis formation, whereas the opposite effects are observed after injection of exogenous hNT‐3 into the I/R hearts at the beginning of reperfusion followed by a 6‐day continuously subcutaneous administration of hNT‐3. The protective effects of exogenous delivery of NT‐3 are observed in mouse models of limb ischemia 26 and in cerebral I/R injury. 46 Thus, NT‐3 is an endogenous protective factor and might act as a novel potential agent for the treatment of ischemic heart diseases.

Thirdly, our results provide the first insight and define the underlying mechanisms of the protective function of NT‐3 in cardiomyocytes subjected to I/R injury. This is supported by the following observations: (i) AMCMs express NT‐3; (ii) myocardial I/R‐induced apoptosis of cardiomyocytes is significantly reduced by upregulation of NT‐3 with exogenous hNT‐3, whereas cardiomyocyte apoptosis increases with downregulation of endogenous NT‐3 with Ad‐shNT‐3 or neutralization of NT‐3 with its neutral antibody in adult mice hearts subjected to I/R injury; and (iii) similar results are further confirmed in sI/R AMCMs. In addition, the angiogenic effects of NT‐3 in I/R hearts are consistent with observations in a murine model of limb ischemia. 25 , 26 Therefore, improved cardiac function and reduced fibrosis formation by implantation of hCVPCs and exogenous hNT‐3 in I/R hearts are at least related to the improvement of cardiomyocyte survival and angiogenesis. As NT‐3 is expressed in cardiac fibroblasts, the regulatory effect of NT‐3 in fibroblasts and its contribution to fibrosis formation in I/R hearts need to be determined in the future. It has been documented that activation of prosurvival kinases (such as Akt and ERK) can achieve powerful cardioprotection when activated at the time of myocardial reperfusion. 4 , 33 , 47 However, activation of these pathways at the time of myocardial reperfusion is not sufficient to confer cardioprotection, and additional stimulus are needed to increase the activation of these pathways to effectively protect the heart against I/R injury. 34 , 48 NT‐3 can activate kinase cascades such as Akt in microvascular endothelial cells of limb, 26 P38 in glioblastoma cells 49 or ERK in Müller cells 50 via binding to TrkC to play a fundamental role in cell survival and proliferation after ischemic injury. 25 Interestingly, we observed that at day 1 post‐reperfusion I/R‐increased phosphorylation of ERK is further enhanced by injection of exogenous hNT‐3 or hCVPCs but not the phosphorylation of AKT and P38, whereas the activation of ERK and the cardioprotective effects of hNT‐3 are abolished by an ERK specific inhibitor, SCH772984. Therefore, activation of the ERK signaling pathway is responsible for the NT‐3‐mediated cardioprotection, including the improvement of cardiomyocyte survival in I/R hearts. This is further supported by observations that enhanced Bim (a directly downstream target of activated ERK 40 ), in I/R hearts is significantly inhibited by hNT‐3, concomitantly with decrease of cCas‐3 in sI/R cardiomyocytes. However, opposite effects are observed in I/R hearts and sI/R cardiomyocytes with SCH772984 treatment. RSK is another direct downstream target of phosphorylated ERK, 41 , 51 which has the opposite role to Bim in apoptosis where RSK inhibiting apoptosis but Bim initiating apoptosis. 40 As phosphorylation of RSK is not altered in I/R hearts with or without hNT‐3 treatment, the ERK‐Bim‐caspase 3 pathway contributes to the NT‐3‐mediated prosurvival effects in cardiomyocytes during the healing process of I/R hearts. Other proteins secreted from hCVPCs could be involved in the modulation of apoptosis in cardiomyocytes and angiogenesis, resulting in promoting of infarct heart healing. It is worthy further study to thoroughly identify potentially secreted‐proteins from hCVPCs which contribute to cardioprotection. These findings, together with the observations of the cardiac benefits from various cell‐based therapies when delivered during the early phase of I/R or MI, 7 , 8 , 15 suggest that the paracrine effects are a critical mechanism in cell‐based therapies for myocardial I/R via triggering of endogenous cardioprotective and repair programs. 52 , 53 Thus, further characterization of the stem cell secretome and identification of specific paracrine factors would provide an opportunity to develop protein therapeutics that protect and repair damaged hearts.

In this study, we showed that the improvement of cardiomyocyte survival and vascularization in the infarcted hearts by hCVPCs and hNT‐3 are associated with the better cardiac function and reduced fibrosis formation. This is consistent with previous reports showing that anti‐apoptosis/necrosis of cardiomyocytes and/or improvement of angiogenesis can significantly reduce scar size in MI or I/R hearts. 54 , 55 Because MI and I/R cause apoptosis/necrosis of cardiomyocytes and destroy capillary networks, to rebuild vascular network after MI or I/R is essential for delivering oxygen and metabolites to cardiac cells to help recovery of damaged cells and thereby links to reduce scar size. 1 , 54 , 56 , 57 , 58 Thus, therapeutic approaches that improve myocyte survival and angiogenesis in infarcted hearts are beneficial for reduction of fibrosis formation.

5. CONCLUSION

We have demonstrated that NT‐3 is a novel endogenous cardioprotective factor secreted from hCVPCs and is enhanced in the I/R heart. It contributes to the delivery of hCVPC‐mediated improvement of cardiac function and limitation of fibrosis formation, and the endogenous NT‐3 in the hearts attenuates myocardial I/R injury too. The beneficial effects of NT‐3 are the result of improvement of cardiomyocyte survival through the ERK‐Bim signaling pathway and the promotion of angiogenesis in I/R hearts. The findings provide novel insights into the role and mechanisms of the neurotrophin family member in the improvement of cardiomyocyte survival in I/R hearts, the paracrine action of hCVPCs in cell therapies for the ischemic heart disease, and the value of cell products in cell‐free approaches for the treatment of ischemic heart disease.

CONFLICT OF INTEREST

The authors declared no potential conflicts of interest.

AUTHOR CONTRIBUTIONS

W.B., J.W.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; Y.J.: provided hCVPCs, performed part experiments of immunofluorescence staining; Q. Li: designed research, isolated some adult mice cardiomyocytes, manuscript writing; S.W.: provided hCVPC‐CdM; M.L.: financial support, collection of in vitro data; Q. Liu: performed the in vitro experiments; proteome analyses of hESC‐CVPC‐conditional medium; F.L.: reagent ordering and other support; C.P.: manuscript revision and language editing; Y.W.: data analysis, manuscript revision; H.T.Y.: conception and design, data analysis and interpretation, manuscript writing, financial support, final approval of manuscript.

Supporting information

Data S1: Supporting Information.

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Figure S1: Harvests and characterizes hCVPCs. A, Differentiated SSEA1+ hCVPCs were assessed with flow cytometry. n = 3. B, Differentiated SSEA1+ hCVPCs were immunostained with the expression of cardiac progenitor cell markers. n = 3. Scale bar = 50 μm. SSEA1, stage‐specific embryonic antigen 1; hCVPCs, human embryonic stem cell‐derived cardiovascular progenitor cells; MESP1, mesoderm posterior BHLH transcription factor 1; ISL1, ISL LIM homeobox 1; MEF2C, myocyte enhancer factor 2C; GATA4, GATA binding protein 4; NKX 2.5, NK2 homeobox 5.

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Figure S2: hCVPCs improves angiogenesis in the infarct and border zones of hearts postischemia/reperfusion (I/R). A, IHC staining and quantitative data for CD31+ endothelial cells in I/R + PM (Puramatrix) group and I/R + hCVPCs group at day 28 post‐I/R. n = 5. Scale bar = 50 μm. B, IHC staining and quantitative data for α‐SMA+ blood vessels in I/R + PM group and I/R + hCVPCs group at day 28 post‐I/R. n = 5. Scale bar = 50 μm. *P < .05, ***P < .001.

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Figure S3: NT‐3 is highly secreted from hCVPCs. A, The fold increase of NT‐3 detected with human antibody array in hCVPC‐conditional medium (CdM) compared with the DMEM group. B, qRT‐PCR analysis of NT‐3 expression in hESCs and hCVPCs. n = 4. C, The concentration of NT‐3 in hCVPC‐CdM analyzed by the ELISA. n = 3. **P < .01, ***P < .001.

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Figure S4: Human recombinant NT‐3 (hNT‐3) has no influence on cardiac function of C57BL/6 mice. Left ventricle (LV) ejection fraction (LVEF), LV fractional shortening (LVFS) and LV systolic dimension (LVDs) measured by echocardiography at day 2, 7, 28 after myocardial injection of hNT‐3 (0.5 μg/20 μL, GeneTex) or PBS (phosphate‐buffered saline, 20 μL) in the LV, followed by continuously releasing hNT‐3 (0.5 μg d−1) or PBS up to 1 week via Micro‐Osmotic Pumps (Model 1004, Alzet) implanted subcutaneously. n = 5. ns, nonsignificant.

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Figure S5: Exogenous NT‐3 partially reversed I/R‐induced myocyte hypertrophy. Representative and quantitative data of FITC‐labeled WGA immunostaining for cardiomyocytes in the same level of LV cross sections from sham or infarcted hearts at 28 days of post‐I/R with and without NT‐3 treatment. n = 5 hearts each. Scale bar = 50 μm. *P < .05, ***P < .001.

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Figure S6: Exogenous NT‐3 decreases level of activated Cas‐3 in vivo. Representative images and averaged Western blot analysis for Cas‐3, cCas‐3 and GAPDH in the infarct and border zones of day 1 post‐I/R hearts between I/R + PBS and I/R + hNT‐3 group compared with Sham mice. n = 3. Cas‐3, caspase‐3; cCas‐3, cleaved caspase‐3; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase. **P < .01, ***P < .001.

Click here for additional data file. (825.8KB, tif)

Figure S7: The expression of NT‐3 in cardiac cells and dynamic change of NT‐3 in hearts after I/R. A, Representative images and averaged Western blot analysis for NT‐3 and GAPDH in the isolated adult mice cardiomyocytes (AMCMs) and cardiac fibroblasts (AMCFs). B, Representative images and averaged Western blot analysis for NT‐3 and GAPDH in the infarct and border zones of day 1 post‐I/R hearts during different time point of I/R compared with Sham mice. n = 3‐4. ***P < .001.

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Figure S8: Downregulation of NT‐3 by short hairpin RNA has no influence on cardiac function of C57BL/6 mice but increased apoptosis after I/R. A, Representative images and averaged Western blot analysis for efficiency of adenovirus down regulation at day 4 postinjection in hearts. n = 3. B, The cardiac function of LVEF, LVFS and LVDs measured by echocardiography at day 4 after recombinant adenoviruses injection. n = 4‐5. C, Representative images and averaged Western blot analysis for Cas‐3, cCas‐3 and GAPDH in the infarct and border zones of day 1 post‐I/R hearts between I/R + Ad‐shNC and I/R + Ad‐shNT‐3 group compared with Sham mice. n = 3‐4. Ad‐shNC, adenovirus‐short hairpin RNA of negative control; Ad‐sh‐NT‐3, adenovirus‐short hairpin RNA of mouse NT‐3. *P < .05, ***P < .001; ns, nonsignificant.

Click here for additional data file. (1.4MB, tif)

Figure S9: hNT‐3‐treated do not enhance phosphorylation level of Akt and P38 at day 1 post‐I/R. A, Representative images and averaged pixel density analysis for P‐ERK, T‐ERK and GAPDH in the infarct and border zones of day 1 post‐I/R hearts between I/R + PBS and I/R + hNT‐3 group. n = 2. B, Representative and averaged Western blot analysis for P‐Akt (The308), P‐Akt (Ser473), T‐Akt, P‐P38, T‐P38 and GAPDH in PBS and hNT‐3 treated mice hearts at 1 day after I/R. n = 3. T‐ERK, total‐extracellular signal‐regulated kinase; P‐ERK, phosphorylation of ERK; Akt/PKB, total‐protein kinase B. *P < .05; ns, nonsignificant.

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Figure S10: Determination of the effective concentration of ERK specific inhibitor (SCH772984) in mice I/R model. 10 μg SCH772984 decreased the level of P‐ERK which stimulated with 0.5 μg hNT‐3 treatment in mice hearts after I/R. n = 3. ns, nonsignificant.

Click here for additional data file. (908.5KB, tif)

Figure S11: SCH772984/SCH effectively blocks NT‐3 stimulated phosphorylation of ERK and increases cell death of AMCMs in vitro. A, Determination of effective dose of SCH in vitro. n = 3. B, Representative images of AMCMs morphology and quantitative percentages of round/total cells after sI/R injury with or without NT‐3 (10 ng/mL) and SCH. n = 3‐4. Scale bar = 200 μm. C, Representative images and averaged Western blot analysis for Bim, Cas‐3, cCas‐3 and GAPDH in AMCMs among sI/R, sI/R + hNT‐3, sI/R + SCH and sI/R + SCH + hNT‐3 groups. n = 3. sI/R, simulated ischemia/reperfusion. *P < .05, **P < .01, ***P < .001; ns, nonsignificant.

Click here for additional data file. (2MB, tif)

Figure S12: Determination the effective concentration of NT‐3 neutralizing antibody (NT‐3‐Ab) in AMCMs sI/R model. 300 ng/mL NT‐3‐Ab abolished the cardioprotection of hCVPC‐CdM for LDH release in AMCMs against sI/R injury. n = 4‐8. *P < .05, ***P < .001; ns, nonsignificant.

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Table S1: Antibody information

Click here for additional data file. (13.3KB, docx)

ACKNOWLEDGMENTS

This work was supported by grants from National Key R&D Program of China (2017YFA0103700 and 2016YFC1301204 to YHT), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA16010201 to YHT), National Natural Science Foundation of China (81520108004 and 81470422 to YHT, 81500235 to LML), and Major Program of Development Fund for Shanghai Zhangjiang National Innovation Demonstration Zone (ZJ2018‐ZD‐004 to YHT) and the Science and Technology Commission of Shanghai Municipality (17431906600 to YHT). The authors thank Mr. Xiujian Yu in Shanghai Institute of Nutrition and Health for the technical support and thank WiCell Research Institute for providing the H9 hESCs.

Bi W, Wang J, Jiang Y, et al. Neurotrophin‐3 contributes to benefits of human embryonic stem cell‐derived cardiovascular progenitor cells against reperfused myocardial infarction. STEM CELLS Transl Med. 2021;10:756–772. 10.1002/sctm.20-0456

Wei Bi and Jinxi Wang contributed equally to this study.

Funding information Science and Technology Commission of Shanghai Municipality; Major Program of Development Fund for Shanghai Zhangjiang National Innovation Demonstration Zone; National Natural Science Foundation of China; Strategic Priority Research Program of the CAS; National Key R&D Program of China

DATA AVAILABILITY STATEMENT

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

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1: Supporting Information.

Click here for additional data file. (195.9KB, pdf)

Figure S1: Harvests and characterizes hCVPCs. A, Differentiated SSEA1+ hCVPCs were assessed with flow cytometry. n = 3. B, Differentiated SSEA1+ hCVPCs were immunostained with the expression of cardiac progenitor cell markers. n = 3. Scale bar = 50 μm. SSEA1, stage‐specific embryonic antigen 1; hCVPCs, human embryonic stem cell‐derived cardiovascular progenitor cells; MESP1, mesoderm posterior BHLH transcription factor 1; ISL1, ISL LIM homeobox 1; MEF2C, myocyte enhancer factor 2C; GATA4, GATA binding protein 4; NKX 2.5, NK2 homeobox 5.

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Figure S2: hCVPCs improves angiogenesis in the infarct and border zones of hearts postischemia/reperfusion (I/R). A, IHC staining and quantitative data for CD31+ endothelial cells in I/R + PM (Puramatrix) group and I/R + hCVPCs group at day 28 post‐I/R. n = 5. Scale bar = 50 μm. B, IHC staining and quantitative data for α‐SMA+ blood vessels in I/R + PM group and I/R + hCVPCs group at day 28 post‐I/R. n = 5. Scale bar = 50 μm. *P < .05, ***P < .001.

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Figure S3: NT‐3 is highly secreted from hCVPCs. A, The fold increase of NT‐3 detected with human antibody array in hCVPC‐conditional medium (CdM) compared with the DMEM group. B, qRT‐PCR analysis of NT‐3 expression in hESCs and hCVPCs. n = 4. C, The concentration of NT‐3 in hCVPC‐CdM analyzed by the ELISA. n = 3. **P < .01, ***P < .001.

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Figure S4: Human recombinant NT‐3 (hNT‐3) has no influence on cardiac function of C57BL/6 mice. Left ventricle (LV) ejection fraction (LVEF), LV fractional shortening (LVFS) and LV systolic dimension (LVDs) measured by echocardiography at day 2, 7, 28 after myocardial injection of hNT‐3 (0.5 μg/20 μL, GeneTex) or PBS (phosphate‐buffered saline, 20 μL) in the LV, followed by continuously releasing hNT‐3 (0.5 μg d−1) or PBS up to 1 week via Micro‐Osmotic Pumps (Model 1004, Alzet) implanted subcutaneously. n = 5. ns, nonsignificant.

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Figure S5: Exogenous NT‐3 partially reversed I/R‐induced myocyte hypertrophy. Representative and quantitative data of FITC‐labeled WGA immunostaining for cardiomyocytes in the same level of LV cross sections from sham or infarcted hearts at 28 days of post‐I/R with and without NT‐3 treatment. n = 5 hearts each. Scale bar = 50 μm. *P < .05, ***P < .001.

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Figure S6: Exogenous NT‐3 decreases level of activated Cas‐3 in vivo. Representative images and averaged Western blot analysis for Cas‐3, cCas‐3 and GAPDH in the infarct and border zones of day 1 post‐I/R hearts between I/R + PBS and I/R + hNT‐3 group compared with Sham mice. n = 3. Cas‐3, caspase‐3; cCas‐3, cleaved caspase‐3; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase. **P < .01, ***P < .001.

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Figure S7: The expression of NT‐3 in cardiac cells and dynamic change of NT‐3 in hearts after I/R. A, Representative images and averaged Western blot analysis for NT‐3 and GAPDH in the isolated adult mice cardiomyocytes (AMCMs) and cardiac fibroblasts (AMCFs). B, Representative images and averaged Western blot analysis for NT‐3 and GAPDH in the infarct and border zones of day 1 post‐I/R hearts during different time point of I/R compared with Sham mice. n = 3‐4. ***P < .001.

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Figure S8: Downregulation of NT‐3 by short hairpin RNA has no influence on cardiac function of C57BL/6 mice but increased apoptosis after I/R. A, Representative images and averaged Western blot analysis for efficiency of adenovirus down regulation at day 4 postinjection in hearts. n = 3. B, The cardiac function of LVEF, LVFS and LVDs measured by echocardiography at day 4 after recombinant adenoviruses injection. n = 4‐5. C, Representative images and averaged Western blot analysis for Cas‐3, cCas‐3 and GAPDH in the infarct and border zones of day 1 post‐I/R hearts between I/R + Ad‐shNC and I/R + Ad‐shNT‐3 group compared with Sham mice. n = 3‐4. Ad‐shNC, adenovirus‐short hairpin RNA of negative control; Ad‐sh‐NT‐3, adenovirus‐short hairpin RNA of mouse NT‐3. *P < .05, ***P < .001; ns, nonsignificant.

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Figure S9: hNT‐3‐treated do not enhance phosphorylation level of Akt and P38 at day 1 post‐I/R. A, Representative images and averaged pixel density analysis for P‐ERK, T‐ERK and GAPDH in the infarct and border zones of day 1 post‐I/R hearts between I/R + PBS and I/R + hNT‐3 group. n = 2. B, Representative and averaged Western blot analysis for P‐Akt (The308), P‐Akt (Ser473), T‐Akt, P‐P38, T‐P38 and GAPDH in PBS and hNT‐3 treated mice hearts at 1 day after I/R. n = 3. T‐ERK, total‐extracellular signal‐regulated kinase; P‐ERK, phosphorylation of ERK; Akt/PKB, total‐protein kinase B. *P < .05; ns, nonsignificant.

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Figure S10: Determination of the effective concentration of ERK specific inhibitor (SCH772984) in mice I/R model. 10 μg SCH772984 decreased the level of P‐ERK which stimulated with 0.5 μg hNT‐3 treatment in mice hearts after I/R. n = 3. ns, nonsignificant.

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Figure S11: SCH772984/SCH effectively blocks NT‐3 stimulated phosphorylation of ERK and increases cell death of AMCMs in vitro. A, Determination of effective dose of SCH in vitro. n = 3. B, Representative images of AMCMs morphology and quantitative percentages of round/total cells after sI/R injury with or without NT‐3 (10 ng/mL) and SCH. n = 3‐4. Scale bar = 200 μm. C, Representative images and averaged Western blot analysis for Bim, Cas‐3, cCas‐3 and GAPDH in AMCMs among sI/R, sI/R + hNT‐3, sI/R + SCH and sI/R + SCH + hNT‐3 groups. n = 3. sI/R, simulated ischemia/reperfusion. *P < .05, **P < .01, ***P < .001; ns, nonsignificant.

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Figure S12: Determination the effective concentration of NT‐3 neutralizing antibody (NT‐3‐Ab) in AMCMs sI/R model. 300 ng/mL NT‐3‐Ab abolished the cardioprotection of hCVPC‐CdM for LDH release in AMCMs against sI/R injury. n = 4‐8. *P < .05, ***P < .001; ns, nonsignificant.

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Table S1: Antibody information

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Data Availability Statement

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

Articles from Stem Cells Translational Medicine are provided here courtesy of Oxford University Press

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