Deficiency of GDF-11 Accelerates TAC-Induced Heart Failure by Impairing Cardiac Angiogenesis

Figures

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  Figure 1. GDF11 Expression Is Upregulated in Stressed Hearts and Is Mainly Produced by CMs (A) Representative Western blot and quantitation of growth differentiation factor (GDF)-11 and sarco(endo)plasmic reticulum calcium adenosine triphosphatase 2a (SERCA2a) protein in the heart from healthy and dilated cardiomyopathy (DCM) patients (n = 12 for DCM; n = 9 for healthy patient GDF-11 level and n = 6 for healthy patient SERCA2a level). (B) The relative levels of GDF-11 messenger RNA (mRNA) in healthy and DCM hearts (n = 12 for DCM patients, n = 9 for healthy patients). Values represent the fold changes relative to the donors. The actin mRNA level was used as the internal control. (C) Representative Western blot and quantitation of GDF-11 protein in mouse hearts after sham surgery and 14, 28, and 56 days after transverse aortic constriction (TAC) (n = 8 for sham, n = 7 for TAC 14 days and TAC 28 days, n = 5 for TAC 56 days). (D) The relative levels of GDF11 mRNA in sham and TAC hearts (n = 3 for sham and TAC). Values represent the fold changes relative to the sham. The actin mRNA level was used as the internal control. (E) Quantification of GDF11 mRNA (normalized to actin mRNA) from…

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  Figure 2. Lack of CM GDF-11 Does Not Affect Cardiac Function (A) (Top) Gross morphology of Cre and cardiomyocyte-specific knockout (CKO) hearts (scale bar = 1 mm). (Bottom) Representative hematoxylin and eosin (H&E) stain of longitudinal sectional heart from Cre and CKO mice (scale bar = 1 mm). (B) For both male and female mice, the body weight was measured in CKO and Cre mice (as control animal) after injection of tamoxifen (Tam) at the indicated days (n = 6 for Cre/CKO at Tam 0-7 days, n = 3 for Cre/CKO at Tam 30 -50 days). (C) Heart weight-to-body weight (HW/BW) ratio in Cre and CKO mice at 12 weeks (n = 10 for Cre, n = 5 for CKO). (D) Representative echocardiographic M-mode images of Cre and CKO hearts. Quantification of the ventricular function reflected by ejection fraction (EF) and fractional shortening (FS) for 4 months (n = 6 for Cre, n = 6 for CKO). (E) Representative H&E stain of transverse cross-section and magnification partial image (scale bar = 50 μm) from Cre and CKO mice at 8 weeks. (F) Representative wheat germ agglutinin (WGA) (scale bar = 50 μm) and Picrosirius Red (PSR) stain (scale bar = 50 μm) images of transverse sections from Cre and CKO hearts. Quantification of…

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  Figure 3. GDF11 Deficiency Accelerates the Transition From Compensated Hypertrophy to Heart Failure (A) Representative images and quantification of echocardiographic FS and left ventricular end-diastolic diameter (LVIDd) of mice treated as described in the indicated groups (n = 8 for baseline [BL], n = 7 for TAC 46 days). (B) Histological analyses of heart sections stained with H&E from the indicated groups after TAC 16 days and 46 days. (C) Gross morphology of Cre and CKO heart after TAC 46 days. Comparison of the HW/BW and heart weight-to-tibial length (HW/TL) ratios in Cre and CKO mice (n = 5 for Cre, n = 7 for CKO). (D) Representative images of WGA (scale bar = 50 μm) from the indicated groups 46 days after TAC. The quantification of average cross-sectional area of CMs in the indicated groups were shown in bar graph (n > 50 cells/group for CM area; n = 13 for Cre, n = 15 for CKO). (E) Interstitial and perivascular Sirius red staining (scale bar = 20 μm) was performed to determine cardiac fibrosis of the hearts from CKO and Cre mice after TAC 46 days. Quantification of cardiac collagen volume in the indicated groups (n = 12 for Cre, n = 10 for CKO). (F) Quantitative…

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  Figure 4. GDF11 Deficiency Causes Decreased Cardiac Angiogenesis and VEGF Expression Following Pressure Overload (A) Representative immunofluorescent images of heart sections from Cre and CKO mice 56 days post-TAC after staining for CD31 (red), troponin I (green), and DAPI (blue). CD31-positive cells were quantified in the bar graph (scale bar = 50 μm) (n = 12). ∗∗∗P < 0.001 vs Cre. (B) Representative image of heart sections stained with α-smooth muscle actin (SMA) (green), troponin I (red), and DAPI (blue), and the quantification of αSMA-positive cells (scale bar = 50 μm) (n = 25). (C) Quantitative reverse-transcriptase polymerase chain reaction was performed to analyze the mRNA levels of vascular endothelial growth factor (VEGF) genes in the post-TAC CKO and Cre mice (n = 7). ∗P < 0.05 vs sham-Cre. #P < 0.05 vs TAC-Cre. (D) Immunoblot for the VEGF proteins from hearts of Cre and CKO mice after sham or TAC surgery at 56 days (n = 7). ∗∗P < 0.01 vs Cre. (E) Schematic diagram of the primary neonatal mouse CM paracrine experiment. The conditioned medium was concentrated 10-fold and then co-cultured with human umbilical vein endothelial cells (HUVECs) for tube formation. Representative images…

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  Figure 5. GDF11 Does Not Induce or Reverse Cardiac Hypertrophy, but Rather an Endogenous Protective Factor (A) Representative images and percentage of Edu-positive CMs (indicated neonatal mouse CMs) after treatment with different doses of recombinant growth differentiation factor (GDF)-11 (25, 50, and 100 mg/μL) (n = 6 for Dulbecco’s modified Eagle medium [DMEM] and 100 mg/μL, n = 12 for 25 mg/μL, n = 8 for 50 mg/μL). (B) Representative images of neonatal rat CMs (treated with 50 mg/μL rGDF11 or not) stained by troponin I (red) and DAPI (blue), and the quantification of cell surface area (scale bar = 50 μm) (n > 10 cells/group; n = 10). (C) Quantification of mRNA levels about cardiac hypertrophic markers (ANP and BNP) in neonatal mouse CMs after treatment with different dose of rGDF-11 (25, 50, and 100 mg/μL) (n = 3 for DMEM and 100 mg/μL, n = 4 for 25 mg/μL and 50 mg/μL). (D) Representative immunoblot and quantitation of SERCA2a and VEGFA proteins from neonatal mouse CMs after treatment with rGDF-11. β-actin was the loading control scenario (n = 6). ∗∗∗P < 0.001 vs CMs. (E) Quantification of mRNA levels about GDF11, cardiac hypertrophic markers (ANP, BNP, β-MHC, SERCA2a), Trim63, and…

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  Figure 6. GDF-11 Alters Akt-mTOR Signaling Pathway Through Binding the Transforming Growth Factor β Type I Receptor in CMs (A) Representative immunoblot and quantification of phosphorylated Akt (p-Akt) and total Akt (T-Akt) and its downstream target mTOR, and SERCA2a levels in the CMs transfected with siRNA (siNC vs siGDF-11) (n = 3).∗P < 0.05 vs siNC. (B) Immunoblot and quantification of SERCA2a, p-Akt, mTOR, FOXO3a, and 4E-BP1 levels in the CMs transfected with the null vector (NC) and LV-GDF-11 (n = 3). ∗P < 0.05 vs NC. (C) Immunoblot and quantification of SERCA2a, p-Smad3/T-Smad3, p-Akt/T-Akt, and VEGFA in CMs transfected with the null vector and LV-GDF-11 followed by treatment with Smad3 inhibitor (SIS3) (n = 3). ∗P < 0.05 vs NC. #P < 0.05 vs NC+SIS3. (D) Immunoblot and quantification of levels of p-Akt/T-Akt, SERCA2a, and VEGFA expression in CMs transfected with null-vector and LV-GDF11 followed by SB431542 (a transforming growth factor-β type I receptor inhibitor) (n = 3). ∗P < 0.05 vs NC. (E) Immunoblot and quantification of levels of p-Akt/T-Akt, p-Smad3/T-Smad3, and VEGFA in CMs transfected the with null vector and LV-GDF-11 followed by treatment with an Akt inhibitor (n = 4). ∗P…

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  Figure 7. GDF-11 Compensated by AAV9 Rescued the Cardiac Dysfunction in Knockout mice (A) Representative images and quantification of echocardiographic FS of mice treated as described in the indicated groups (n = 5). (B) Representative images of WGA (scale bar = 50 μm) from the indicated groups after TAC. The quantification of average cross-sectional area of CMs in the indicated groups are shown in bar graph (n = 20). (C) Representative images of CD31 (red) from the indicated groups after TAC. The quantification of capillaries per CM in the indicated groups is shown in bar graph (n = 15). (D) Histological analyses of heart sections stained with Sirius red (scale bar = 100 μm) from the indicated groups after TAC (n = 15). ∗∗P < 0.01, ∗∗∗P < 0.001 vs Cre-Vec. #P < 0.05, ##P < 0.01 vs Cre-gdf11. AAV9 = adeno-associated virus serotype 9; TNI = troponin I; other abbreviations as in Figures 1, 2, and 4.