Journal: Cardiovascular Research

Article Title: Circulating GDF11 exacerbates myocardial injury in mice and associates with increased infarct size in humans

doi: 10.1093/cvr/cvad153

Figure Lengend Snippet: Age-mediated and myocardial I/R injury–mediated changes in Gdf11 and Mstn expression (top) and experimental study design (bottom). ( A ) Age-dependent changes in myocardial Gdf11 and Mstn mRNA expression levels ( n = 5/group). ( B ) Myocardial mRNA expression of Gdf11 and Mstn of aged mice subjected to I/R injury or respective controls ( n = 5/group). ( C ) Experimental scheme. Young (3–4 months) and aged (22–24 months) C57BL/6 mice were randomized to receiving rGDF11 (0.1 mg/kg BW daily i.p.) or vehicle (CTRL) over 30 days, as reported previously. Following I/R injury, morphological analyses and targeted transcriptomics were performed. P values were calculated by one-way ANOVA ( A ) or unpaired Student’s t -test ( B ). Data in A and B are presented as violin plots (median and IQR) with single data points superimposed.

Article Snippet: Upon reviewing of these results ( n = 5 per group), both young (3–4 months) and aged (22–24 months) mice were assigned to receive daily i.p. injections of either human rGDF11 [0.1 mg/kg body weight (BW); PeproTech, disulfide-linked homodimer comprising two 109 amino acid polypeptide chains; online, ] or volume-adjusted vehicle [0.1% bovine serum albumin (BSA) containing phosphate buffered saline (PBS)] over 30 days, a duration necessary to achieve significant elevations in vivo .

Techniques: Expressing

Journal: Cardiovascular Research

Article Title: Circulating GDF11 exacerbates myocardial injury in mice and associates with increased infarct size in humans

doi: 10.1093/cvr/cvad153

Figure Lengend Snippet: LC/LC-MS-based determination of circulating Gdf11 (left) and Mstn (right) upon rGDF11 delivery. ( A ) Daily injections of rGDF11 (0.1 mg/kg BW i.p./day; n = 11–16 mice/group) lead to a consistent increase in circulating Gdf11 after 7 days, as suggested by a previous report, and now, for the first time, confirmed by LC/LC-MS. ( B ) Importantly, circulating Mstn levels remain unaffected by rGDF11 supplementation. P values were calculated by two-way ANOVA. Data are shown as mean and SEM for each timepoint.

Article Snippet: Upon reviewing of these results ( n = 5 per group), both young (3–4 months) and aged (22–24 months) mice were assigned to receive daily i.p. injections of either human rGDF11 [0.1 mg/kg body weight (BW); PeproTech, disulfide-linked homodimer comprising two 109 amino acid polypeptide chains; online, ] or volume-adjusted vehicle [0.1% bovine serum albumin (BSA) containing phosphate buffered saline (PBS)] over 30 days, a duration necessary to achieve significant elevations in vivo .

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Cardiovascular Research

Article Title: Circulating GDF11 exacerbates myocardial injury in mice and associates with increased infarct size in humans

doi: 10.1093/cvr/cvad153

Figure Lengend Snippet: Histological and biochemical abnormalities of mice treated with rGDF11 or vehicle and subjected to myocardial I/R injury stratified by age. ( A , D ) Infarct size (I) and area at risk (AAR) relative to ventricle surface (calculated as I/V and AAR/V, respectively). cTnI denotes cardiac troponin I. Representative images of triphenyltetrazolium chloride-stained middle heart sections of vehicle- vs. rGDF11-treated mice are shown. ( B , E ) Quantification of neutrophil and macrophage infiltration together with representative histology (top) and CXCL1 and CCL2 serum levels (bottom) following acute myocardial I/R injury. ( C , F ) DiBrY- and 4-HNE-positive areas in tissue sections of infarcted hearts of both groups. P values were calculated by unpaired Student’s t -test ( A–F ). Data are presented as bar graphs and error bars (mean and SEM) with single data points superimposed.

Article Snippet: Upon reviewing of these results ( n = 5 per group), both young (3–4 months) and aged (22–24 months) mice were assigned to receive daily i.p. injections of either human rGDF11 [0.1 mg/kg body weight (BW); PeproTech, disulfide-linked homodimer comprising two 109 amino acid polypeptide chains; online, ] or volume-adjusted vehicle [0.1% bovine serum albumin (BSA) containing phosphate buffered saline (PBS)] over 30 days, a duration necessary to achieve significant elevations in vivo .

Techniques: Staining

Journal: Cardiovascular Research

Article Title: Circulating GDF11 exacerbates myocardial injury in mice and associates with increased infarct size in humans

doi: 10.1093/cvr/cvad153

Figure Lengend Snippet: Systemic GDF11 restoration accelerates cardiomyocyte apoptosis with IPA-guided transcriptomics and immunostaining pointing towards a possible indirect effect mediated by non-myocyte cells. ( A , B ) Number of TUNEL-positive cardiomyocytes in young (3–4 months) and aged (22–24 months) mice. Representative images (×20) are shown on the right of each panel. ( C ) Left: heatmap represents Z -scored cardiac expression of IPA-identified genes in mice subjected to I/R injury and pre-treated with vehicle (CTRL) or rGDF11 (0.1 mg/kg BW i.p./day) over 30 days; right: volcano plot showing gene expression of IPA-identified candidates between both groups. ( D ) Semi-quantitative IHC shows attenuated Nkx2-5 protein expression in rGDF11 pre-treated mice. Scale bars: 2 mm (left panel), 100 μm (right panel). P values were calculated by ( A , B ) unpaired Student’s t -test or ( C ) multiple unpaired two-sample t -tests with Welch’s correction. Data ( A , B , D ) are presented as bar graphs and error bars (mean and SEM) with single data points superimposed.

Article Snippet: Upon reviewing of these results ( n = 5 per group), both young (3–4 months) and aged (22–24 months) mice were assigned to receive daily i.p. injections of either human rGDF11 [0.1 mg/kg body weight (BW); PeproTech, disulfide-linked homodimer comprising two 109 amino acid polypeptide chains; online, ] or volume-adjusted vehicle [0.1% bovine serum albumin (BSA) containing phosphate buffered saline (PBS)] over 30 days, a duration necessary to achieve significant elevations in vivo .

Techniques: Immunostaining, TUNEL Assay, Expressing, Gene Expression

Journal: Skeletal Muscle

Article Title: A GDF11/myostatin inhibitor, GDF11 propeptide-Fc, increases skeletal muscle mass and improves muscle strength in dystrophic mdx mice

doi: 10.1186/s13395-019-0197-y

Figure Lengend Snippet: GDF11PRO-Fc associates with GDF11 and MSTN. Protein-protein interactions between GDF11PRO-Fc or MPRO-Fc and rGDF11, rMSTN, or rActivin A were determined by a pull-down assay. GDF11PRO-Fc or MPRO-Fc was incubated with rGDF11, rMSTN, or rActivin A for 1 h at 4 °C. Fc-fused protein complexes were separated on a protein A/G-coated agarose resin and eluates were run on a 12% SDS-PAGE gel under reducing conditions and probed by western blot. Input control was 5% of the input material. WB western blot

Article Snippet: To assess complex formation, rGDF11 (1958-GD-010; R&D Systems; Minneapolis, MN), recombinant MSTN (rMSTN; 788-G8–010; R&D Systems; Minneapolis, MN) or recombinant activin A (rActivinA; 338-AC-010; R&D Systems; Minneapolis, MN) were added to cell lysates to a final concentration of 100–500 ng/ml.

Techniques: Protein-Protein interactions, Pull Down Assay, Incubation, SDS Page, Western Blot, Control

Journal: Skeletal Muscle

Article Title: A GDF11/myostatin inhibitor, GDF11 propeptide-Fc, increases skeletal muscle mass and improves muscle strength in dystrophic mdx mice

doi: 10.1186/s13395-019-0197-y

Figure Lengend Snippet: GDF11PRO-Fc blocks GDF11/MSTN-induced myotube atrophy in C2C12 cells. a Schematic detailing experimental timeline in C2C12 myotubes. AAV6-EGFP or AAV6-GDF11PRO-Fc was added to C2C12 myotubes at a MOI of 10 on day 5 post-differentiation and 100 ng/ml rGDF11 or rMSTN was added on day 7. Myotubes were stained and analyzed on day 10. b EGFP expression was evident at 48–72 h in C2C12 myotubes treated with AAV6-EGFP (MOI 10 ). Scale bars represents 50 μm. c Vector genome copy number per diploid genome in C2C12 myotubes 72 h after addition of AAV6-EGFP or AAV6-GDF11PRO-Fc (MOI 10 ). d Representative immunofluorescence images of C2C12 myotubes. C2C12 myotube membranes were visualized by staining with an anti-dystrophin antibody (red). Nuclei were stained with DAPI (blue). Inset shows a zoomed-in region. Scale bars represent 50 μm (main panel) and 25 μm (panel inset). e The fraction of nuclei incorporated into myotubes (differentiation index) was calculated and presented as a percentage of control. f Average myotube diameter relative to control and ( g ) distribution of diameter measurements. For myotube diameter measurements, each myotube was measured at three points along the length of the myotube and averaged. h Number of nuclei incorporated per myotube. A minimum of 50 myotubes were analyzed per experimental condition. i pSMAD2/3 relative to tSMAD2/3 was assessed by western blot. Equal protein loading was verified by Ponceau S staining and GAPDH was used as a loading control. Data represents results from three separate experiments. All error bars represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. not significant; compared to AAV6-EGFP-treated control. † p < 0.05; †† p < 0.01; ††† p < 0.001; compared to AAV6-EGFP + ligand-treated. pSMAD2/3: phosphorylated SMAD2/3; tSMAD2/3: total SMAD2/3

Article Snippet: To assess complex formation, rGDF11 (1958-GD-010; R&D Systems; Minneapolis, MN), recombinant MSTN (rMSTN; 788-G8–010; R&D Systems; Minneapolis, MN) or recombinant activin A (rActivinA; 338-AC-010; R&D Systems; Minneapolis, MN) were added to cell lysates to a final concentration of 100–500 ng/ml.

Techniques: Staining, Expressing, Plasmid Preparation, Immunofluorescence, Control, Western Blot