Activation of NRG1-ERBB4 signaling potentiates mesenchymal stem cell-mediated myocardial repairs following myocardial infarction

Abstract

Mesenchymal stem cell (MSC) transplantation has achieved only modest success in the treatment of ischemic heart disease owing to poor cell viability in the diseased microenvironment. Activation of the NRG1 (neuregulin1)-ERBB4 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4) signaling pathway has been shown to stimulate mature cardiomyocyte cell cycle re-entry and cell division. In this connection, we aimed to determine whether overexpression of ERBB4 in MSCs can enhance their cardio-protective effects following myocardial infarction. NRG1, MSCs or MSC-ERBB4 (MSC with ERBB4 overexpression), were transplanted into mice following myocardial infarction. Superior to that of MSCs and solely NRG1, MSC-ERBB4 transplantation significantly preserved heart functions accompanied with reduced infarct size, enhanced cardiomyocyte division and less apoptosis during early phase of infarction. The transduction of ERBB4 into MSCs indeed increased cell mobility and apoptotic resistance under hypoxic and glucose-deprived conditions via a PI3K/Akt signaling pathway in the presence of NRG1. Unexpectedly, introduction of ERBB4 into MSC in turn potentiates NRG1 synthesis and secretion, thus forming a novel NRG1-ERBB4-NRG1 autocrine loop. Conditioned medium of MSC-ERBB4 containing elevated NRG1, promoted cardiomyocyte growth and division, whereas neutralization of NRG1 blunted this proliferation. These findings collectively suggest that ERBB4 overexpression potentiates MSC survival in the infarcted heart, enhances NRG1 generation to restore declining NRG1 in the infarcted region and stimulates cardiomyocyte division. ERBB4 has an important role in MSC-mediated myocardial repairs.

Figure 1

Exogenous ERBB4 was successfully transduced in MSCs. (a) RT-RCR screening of NRG1-ERBB expression in MSCs. MSC expressed NRG1 and ERBB2, but not ERBB4. (b) MSCs were successfully transduced with pGFP or pER4-GFP (map shown in Supplementary Figure 2), confirmed by using a GFP-positive signal detected under a fluorescent microscope (b1). RT-PCR (b2) and western blotting (b3) confirmed successful manipulation of ERBB4 into MSCs. Elevated p-ERBB4 occurred when additional NRG1 was added. p-ERBB4, phosphorylated ERBB4. (c) Lentiviral-transduced MSCe and MSC-ERBB4 possessed multi-lineage differentiation capacity, confirmed by staining (c1) and lineage specific gene expression (c2). (d) Neither MSCe nor MSC-ERBB4 injection raised malignant formation during 8 weeks of observation, with mouse embryonic stem cells as positive control. mESCs, mouse embryonic stem cells. n=4 for each group

Figure 2

Transplantation of MSC-ERBB4 reduced fibrosis and improved heart function. Heart function was measured using a pressure-volume conductance catheter system 4 weeks after injection of NRG1, MSCe or MSC-ERBB4. (a) Representative single PV-loop recording images are shown. MI resulted in a rightward shift in the loop, which increased the volume and depression of the ESPVR slope (aii versus ai, red line). MSC-ERBB4 but not MSCe (av versus aiv) or NRG1(av versus aiii) reduced the volume and restored the original slope of the ESPVR. (b) Enhanced peak velocities of pressure change (dp/dt) occurred during isovolumic contraction (+dp/dt) and isovolumic relaxation (−dp/dt) in mice injected with MSC-ERBB4. (c) ERBB4 overexpression reduced scar formation following MI. Masson's trichrome staining indicated the thinning and expansion of the infarct scar (blue color) in the MI group (cii versus ci) and was attenuated in the MSC and NRG1 treated groups at 4 weeks post-MI (ciii, civ and cv). Representative photomicrographs for each group are shown. (d) Quantification of fibrotic area presented as the percentage of LV area positively stained with Masson trichrome. (e) Quantification of LV wall thickness. MSC-ERBB4 transplantation resulted in smaller infarct zone within the total LV area compared with that in the MI, MI+NRG1 and MI+MSCe groups and increased infarct wall thickness. LV, left ventricular; ESPVR, end-systolic pressure-volume relationship. n=12 for each group

Figure 3

ERBB4 overexpression enhanced MSC survival through enhancing the mobility and anti-apoptosis by activating the PI3K/Akt pathway. (a) Surviving MSCe and MSC-ERBB4 were detected using anti-GFP antibody at 4 weeks post-MI. Representative images are shown. Quantification revealed that ERBB4 overexpression significantly improved MSC survival. Scale bar=200 μm. n=12 for each group. (b) The NRG1-ERBB4 pathway enhanced MSC mobility. (b1) Experiment setting: MSCe or MSC-ERBB4 were seeded in the upper chamber of a transwell unit, with NRG1 added to the lower chamber to serve as an attractor. (b2) After hypoxic exposure for 6 hours, MSCe and MSC-ERBB4 exhibited equivalent movements (b2i, NS); however, MSC-ERBB4 demonstrated more aggressive mobility in the presence of NRG1 than MSCe did (b2ii, P<0.05 versus MSCe). Scale bar=200 μm. (c) Annexin V/PI staining followed by flow cytometry were conducted to study the dynamic apoptotic rates of MSCe and MSC-ERBB4. Under hypoxia challenge, MSCe stayed incooperative to NRG1 (cii–cv), but the apoptotic rate of MSC-ERBB4 was attenuated by NRG1 in a dose-dependent manner under hypoxic condition (cvii–cx). The experiment was repeated three times and statistical analysis was performed (cxi). (d) Overexpressing ERBB4 in MSC reduced hypoxia-related apoptosis through the PI3K/Akt pathway. Additional NRG1 increased p-Akt accumulation in MSC-ERBB4 (dviii versus vii), but not in MSCe (dii versus di), and this effect could be partially blocked either by PI3K/Akt inhibitor LY294002 (dxii versus dviii), or anti-ER4 antibody (dxi versus dviii). The same trend was exhibited in Bcl-2 expression. The aforementioned western blotting was repeated three times and representative images are shown. Bcl-2, B-cell lymphoma 2; p-Akt, phosphorylated Akt; t-Akt, total Akt; anti-ERBB4, anti-ERBB4 antibody

Figure 4

ERBB4 overexpression in MSCs provoked cardiomyocyte division and reduced cardiomyocyte loss. (a) Mature cardiomyocyte marker α-actinin and proliferation marker Ki67 indicated that cardiomyocytes underwent mitosis. Both MSCe and MSC-ERBB4 transplantation-stimulated cardiomyocyte proliferation in remote and adjacent areas. Representative images for each group are shown. Quantification indicated that MSC-ERBB4 induced more cardiomyocyte division than MSCe. Scale bar=200 μm. n=8 for each group. (b) TUNEL-labeled apoptotic cardiomyocytes at 48 h after MI. Representative images are shown. MSC-ERBB4 transplantation provided better protection of cardiomyocytes against apoptosis than MSCe did. n=8 for each group

Figure 5

Conditioned medium of MSC-ERBB4 protects cardiomyocytes. (a) The conditioned medium of MSC-ERBB4 promoted cardiomyocyte growth, which was blunted by anti-NRG1 antibody. (b) More Ki67^pos cells were found in α-actinin^pos cardiomyocytes cultured with conditioned medium of MSC-ERBB4 than that of MSCe, indicating a more prosper cell dividing. The number of Ki67^pos cells declined when anti-NRG1 antibody was applied into conditioned medium of MSC-ERBB4. Scale bar=200 μm. (c) The conditioned medium of MSC-ERBB4 prevented cardiomyocytes from senescence, which was erased by anti-NRG1 antibody. (d) The conditioned medium of MSC-ERBB4 prevented cardiomyocytes from apoptosis by enhancing p-Akt and Bcl-2 expression. DAPI, 4',6-diamidino-2-phenylindole; CM, conditioned medium; Bcl-2, B-cell lymphoma 2; p-Akt, phosphorylated Akt; t-Akt, total Akt; anti-NRG1, anti-NRG1 antibody

Figure 5

Conditioned medium of MSC-ERBB4 protects cardiomyocytes. (a) The conditioned medium of MSC-ERBB4 promoted cardiomyocyte growth, which was blunted by anti-NRG1 antibody. (b) More Ki67^pos cells were found in α-actinin^pos cardiomyocytes cultured with conditioned medium of MSC-ERBB4 than that of MSCe, indicating a more prosper cell dividing. The number of Ki67^pos cells declined when anti-NRG1 antibody was applied into conditioned medium of MSC-ERBB4. Scale bar=200 μm. (c) The conditioned medium of MSC-ERBB4 prevented cardiomyocytes from senescence, which was erased by anti-NRG1 antibody. (d) The conditioned medium of MSC-ERBB4 prevented cardiomyocytes from apoptosis by enhancing p-Akt and Bcl-2 expression. DAPI, 4',6-diamidino-2-phenylindole; CM, conditioned medium; Bcl-2, B-cell lymphoma 2; p-Akt, phosphorylated Akt; t-Akt, total Akt; anti-NRG1, anti-NRG1 antibody

Figure 6

Overexpression of ERBB4 in MSCs unexpectedly activates a novel NRG1-ERBB4-NRG1 autocrine loop. Overexpressing ERBB4 in MSCs unexpectedly upregulated the synthesize and secretion of its ligand NRG1, detected by conducting western blotting (a) and ELISA (b), respectively. (c) Overexpressing ERBB4 in MSCs upregulated NRG1, but did not alter expression of ERBB2. ERBB3 remained negative before and after ERBB4 overexpressing. (d) Multiple transient transfections using pER4-GFP confirmed that upregulated NRG1 was attributed to exogenous ERBB4 overexpression. (d1) With increased transfection frequency shown by encircular numbers (d1), GFP signal, which indicated transfection efficiency, was escalating accordingly, from 1.2% for the first time (d1) to 32.7% for the fifth time (d1). (d2) The expression of NRG1 was upregulated along with increased transfection efficiency. (e) In human 293FT cells, NRG1 expression was upregulated after ERBB4 transduction. The aforementioned experiments were repeated three times and representative images are shown. 293FTe, 293FT expressing pGFP; 293FT-ERBB4, 293FT expressing pER4-GFP

Figure 6

Overexpression of ERBB4 in MSCs unexpectedly activates a novel NRG1-ERBB4-NRG1 autocrine loop. Overexpressing ERBB4 in MSCs unexpectedly upregulated the synthesize and secretion of its ligand NRG1, detected by conducting western blotting (a) and ELISA (b), respectively. (c) Overexpressing ERBB4 in MSCs upregulated NRG1, but did not alter expression of ERBB2. ERBB3 remained negative before and after ERBB4 overexpressing. (d) Multiple transient transfections using pER4-GFP confirmed that upregulated NRG1 was attributed to exogenous ERBB4 overexpression. (d1) With increased transfection frequency shown by encircular numbers (d1), GFP signal, which indicated transfection efficiency, was escalating accordingly, from 1.2% for the first time (d1) to 32.7% for the fifth time (d1). (d2) The expression of NRG1 was upregulated along with increased transfection efficiency. (e) In human 293FT cells, NRG1 expression was upregulated after ERBB4 transduction. The aforementioned experiments were repeated three times and representative images are shown. 293FTe, 293FT expressing pGFP; 293FT-ERBB4, 293FT expressing pER4-GFP

Figure 7

Transplantation of MSC-ERBB4 restores reduced NRG1 in MI region. The model of MI was achieved by LAD ligation, resulting in decreasing NRG1 in the infarct region. Neither MSCe nor NRG1 injection improved the situation, but MSC-ERBB4 transplantation restored NRG1 to a level comparable with that of the normal myocardium

Figure 8

Schematic diagram of the NRG1-ERBB4-NRG1 autocrine pathway. Genetic manipulation of ERBB4 into MSC provides irreplaceable partner to ERBB2, the latter of which forms an ERBB2/ERBB4 heterodimer that binds to its ligand NRG1. ERBB4 can bind to NRG1 by forming a homodimer. Activation of the NRG1-ERBB pathway in MSC-ERBB4 enhances cell mobility and anti-apoptosis through the phosphorylation of Akt. Overexpressing ERBB4 in turn regulates the synthesis and secretion of NRG1. The released NRG1 contributes to restore the declined NRG1 level in the infarcted region and support cell growth, dividing and anti-apoptosis of cardiomyocytes. By ERBB4 overexpression in MSCs, we figured out a novel approach that benefits both MSCs and cardiomyocytes, and enable an effective myocardial repair after ischemia