GDF11 enhances therapeutic efficacy of mesenchymal stem cells for myocardial infarction via YME1L-mediated OPA1 processing

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  FIGURE 1. GDF11 protected MSCs against apoptosis under hypoxia condition in vitro. A, GDF11 expression in MSCs were detected by Western blot under normoxia and hypoxia condition at specified times, and β‐actin served as a loading control. B, Quantification of relative GDF11 protein level (n = 3). C, MSCs were pretreated with rGDF11 or overexpressed GDF11 by viral transduction (LV‐GDF11). Representative TUNEL staining images of Control (Ctrl), rGDF11, LV (vector control) and LV‐GDF11 were captured. Scale bar = 50 μm. Quantification of apoptotic cells was presented as ratio of TUNEL‐positive nuclei over the total nuclei from 8 to 10 randomly selected fields in each sample. D, Cleaved caspase 3 (Cl‐cas3) and cleaved caspase 9 (Cl‐cas9) proteins in MSCs pretreated with rGDF11 were assessed by Western blot and quantified by densitometry (n = 3). E, Representative TUNEL staining images of MSCs transfected with siRNA GDF11 (si‐GDF11) or siRNA control (si‐NC). Scale bar = 50 μm. Quantification of apoptotic cells was presented as ratio of TUNEL‐positive nuclei over the total nuclei from 8 to 10 randomly selected fields in each sample. F, Cleaved caspase 3 and 9 proteins of MSCs after transfected…

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  FIGURE 2. GDF11 enhanced paracrine effects of MSCs in vitro. A, Schematic working showed the experiments for evaluating the paracrine effect of MSCs with different treatment. B‐D, The VEGFA levels in conditioned medium from MSCs (Ctrl, rGDF11) (B) or (LV, LV‐GDF11) (C) and (si‐NC, si‐GDF11) (D) were determined by ELISA assay. E, Representative images of tube formation assay of HUVECs/GFP under fluorescent microscopy. HUVECs were cultured with DMEM or conditioned medium of MSCs that had been cultured in the absence (Control) or presence of rGDF11. Scale bar = 100 μm. Quantification of tube formation was shown in bar graphs (n = 8). F, Representative images of tube formation assay of HUVECs/GFP that were cultured with DMEM or conditioned medium of MSCs that had been transfected with siRNAs (si‐GDF11, and NC as negative control). Quantification of tube formation was shown in bar graphs (n = 13). Scale bar = 100 μm. G, TUNEL assay of apoptosis of H9C2 cells that were cultured with DMEM medium or conditioned medium of MSCs that had been cultured in the absence (Control) or presence of rGDF11 (n = 8). Scale bar =50 μm. H, Western blot analysis of apoptosis‐related proteins in H9C2 cells cultured…

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  FIGURE 3. GDF11 protected mitochondrial morphology and function under hypoxic condition. A, Representative images of MSCs under hypoxia condition were taken by electron microscopy (×10 000). Scale bar =1 μm. B, Mitochondrial morphology was analyzed by quantification of area and longitudinal length of mitochondria in MSCsCtrl and MSCsrGDF11 (n = 77 for MSCsCtrl, and n = 95 for MSCsrGDF11). C, D, Mitochondrial respiration reflected by OCR was detected in MSCs alone, MSCs treated with rGDF11 under normoxic (C) or hypoxic (D) conditions. OCRs were assayed under both basal and maximal conditions (n = 3). E. Cellular ATP levels of MSCsCtrl and MSCsrGDF11 under normoxic or hypoxic conditions were measured through luciferin/luciferase‐based assay, and the data were calibrated with protein content. F, Images of MSCs stained with TMRM under fluorescence microscope were used to measure mitochondrial membrane potential. MSCs were treated with rGDF11 (50 ng/mL) for 24 hours then exposed to hypoxia conditions for 48 hours. MSCs treated with either FCCP (50 μmol/L) or oligomycin (10 μmol/L) were served as negative and positive controls, respectively. Scale bar = 100 μm. Bar graph shows relative mean of…

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  FIGURE 4. OPA1 was indispensable for the protective effects of GDF11 in response to hypoxic condition. A, Quantitative analysis of mRNAs of genes involved in mitochondrial homeostasis for MSCsCtrl and MSCsrGDF11 under normoxic or hypoxic condition. Relative mRNA levels were all compared with 18 seconds RNA. B, Western blot analysis of mitochondrial proteins: PGC‐1α, OPA1, Mfn1/2 and DRP1. Monoclonal antibody for OPA1 was used. Quantification of the proteins relative to control β‐actin was shown in right (n = 3). C, Western blot analysis of OPA1, YME1L and OMA1 in MSCsCtrl and MSCsrGDF11 under hypoxic condition. OPA1 detected by a polyclonal antibody. Quantification of the proteins relative to control β‐actin was shown in the bar graph (n = 3). D, Western blot analysis of OPA1 and YME1L in MSCssi‐NC and MSCssi‐GDF11 in the same way as in C (n = 4). E, Western blot analysis of Cyto‐C in cytoplasm and mitochondrial. The values of Cyto‐C in mitochondrial were normalized with TOM20. And the values of Cyto‐C in cytoplasm were normalized with tubulin. Quantification was shown in the bar graph (n = 4). F, TUNEL staining of MSCs that were transfected with siRNAs (si‐NC and si‐OPA1), followed by…

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  FIGURE 5. GDF11‐mediated OPA1 processing depends on YME1L. A, Representative TEM images of MSCs transfected with siRNA‐OPA1 or siRNA‐YME1L for 48 hours and then incubated with rGDF11 (50 ng/mL) for 24 hours and then exposed to hypoxia conditions for 48 hours (×10 000). Scale bars: 1 μm. B, C, Quantification of mitochondrial area, longitudinal length and size distribution according to their length: long tubules (>0.65 μm), intermediate (≤0.65 μm, ≥0.32 μm), and fragmented (<0.32 μm) (n = 105 for MSCsi‐NC, n = 64 for MSCssi‐NC + rGDF11, n = 123 for MSCsi‐OPA1, n = 68 for MSCsi‐OPA1 + rGDF11, n = 93 for MSCsi‐YME1L, and n = 96 for MSCsi‐YME1L + rGDF11). D, Representative immunoblots and densitometric quantification for the expression of YME1L and OMA1 under normoxic and hypoxic conditions (n = 4). E, mRNA levels of YME1L and OMA1 in MSCsCtrl and MSCsrGDF11, and 18 seconds served as control. F, Western blot analysis of YME1L and OPA1. YME1L and L‐OPA1 were quantified and presented as relative level by comparing with β‐actin control (n = 3). G, Intracellular ATP levels in MSCs at specified conditions were determined. ATP levels was calibrated with protein content (n = 3). Data were shown as…

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  FIGURE 6. GDF11 protected MSCs from hypoxia‐induced apoptosis through ALK5‐Smad2/3 pathway. A, Localization of p‐Smad3 in MSCs. DAPI were stained with for nuclear (blue) and fluorescence‐labeled Ab against phosphorylated Smad3 (red). Scale bar = 50 μm. Fluorescence intensity was quantified. B, C, Western blot analysis of p‐Smad3, cleaved caspase 3 and 9 in MSCs which were treated with TGFβR1 inhibitor SB4431542 (B) or p‐Smad3 inhibitor SIS3 (C) for 30 minutes, and incubated with rGDF11 (50 ng/mL) for 24 hours and then exposed to hypoxia condition for 48 hours (n = 3 in B and C). D, Representative TEM images of MSCs treated as described in (B, C) (×10 000). Scale bar =1 μm. Mitochondria were visually scored for their area and longitudinal length. n = 149 for MSCCtrl, n = 63 for MSCsrGDF11, n = 153 for MSCCtrl + SB, n = 160 for MSCrGDF11 + SB, n = 151 for MSCCtrl + SIS and n = 139 for MSC rGDF11 + SIS. E, F, Immunoblot analysis of L‐OPA1 and YME1L in MSCs treated as specified. Relative proteins were presented by comparing each band density with that of β‐actin (n = 3 in E and F). G, H, The abilities of Smad2/3 binding to YME1L promoter sites at 473 to 485 and 2550 to 2562 were analyzed by…

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  FIGURE 7. Delivery of GDF11 overexpressing MSCs improved cardiac function after MI in vivo. A, Representative echocardiographic M‐mode images showed the changes of cardiac function in each group at 28 days after MI. B, Left ventricular ejection fraction and left ventricular fractional shortening were quantified at 3, 7, 14, and 28 days post MI (n = 5 for Sham group, n = 6 for DMEM group, n = 6 for MSCsLV group, and n = 6 for MSCsLV‐GDF11 group, respectively). C, MSCs retention at day 3 post‐MI (n = 6 for each group). Representative images of GFP staining were assayed by fluorescence microscopy, wherein GFP‐positive cells (in green) were specifically counted for MSCs retention, with nuclei stained with DAPI. Scale bar = 100 μm. D, Representative immunofluorescence image of CD31 staining for capillaries (red) and α‐SMA staining for arterioles (green) with nuclei stained with DAPI in the remote zone of ischemic heart from DMEM, MSCsLV, and MSCsLV‐GDF11 group mice at day 28 post‐MI. The capillary and arteriolar densities in the remote zones are quantified by 6‐8 HPFs per section in the bar graphs. Scale bar =100 μm. Data are shown as mean ± SD. *P < .05 vs DMEM/MSCsLV, # P < .05 vs MSCsLV