Gdf11

EYFP-NG2 knock-in mice were implanted intraperitoneally with osmotic minipumps (Alzet Micro-Osmotic Pumps, model 1004, DURECT Corporation) at P150 for continuous i.p. delivery at a flow rate of 0.11 μl/h containing GDF11 (0.1mg/kg/d; rGDF11, catalog # 120-11, PeproTech) or saline. After 4-weeks delivery mice were used for electrophysiological recordings, only cells responding to an agonist were used for comparisons, the proportion of cells responding did not differ between conditions. The use of minipumps ensured reduced stress responses of the animals and consistent dosing compared to i.p. injections which require daily handling.

Recombinant human IL-37 was purchased from Life Technologies (Thermo Fisher Scientific, Gaithersburg, MD) or R&D Systems (Minneapolis, MN). Possible TGF-β1 contamination in IL-37 was undetectable as determined by ELISA (R&D Systems, Minneapolis, MN). Anti-IL-37 antibodies were purchased from R&D Systems (Minneapolis, MN) or Abcam (Cambridge, MA). For Western blot analysis, antibodies against p-Smad1/5/8, Smad1/5/8, p-Smad2/3, Smad2/3 and β-actin were purchased from Cell Signaling Technology. Recombinant human TGF-β, BMP10 and GDF11 were purchased from Peprotech. Anti-human ALK1 antibodies, anti-human ALK5 antibodies, anti-mouse ALK1 antibodies, anti-mouse PECAM antibodies anti-human TGF-β and anti-mouse TGF-β antibodies were purchased from R&D Systems (Minneapolis, MN). ALK1 inhibitor LDN193189 was purchased from Sigma (St. Louis, MO). PE-conjugated streptavidin was purchased from Biolegend (San Diego, CA). Dylight549-conjugated streptavidin was purchased from Abcam (Cambridge, MA). Recombinant soluble chimeric protein containing the ectodomain of human ALK1 conjugated with IgG Fc (ALK1-Fc), chimeric protein containing the ectodomain of human ALK5 conjugated with IgG Fc (ALK5-Fc) and chimeric protein containing ectodomain of human TβRII conjugated with IgG Fc (TβRII-Fc) were purchased from Sinobiological (Beijing, China).

GDF11 (Peprotech, 120-11) was dissolved in water, further diluted according to the manufacturer’s instructions and injected at a concentration of 1 mg kg⁻¹, as previously described in^{24 (link)}. Control mice (young or aged) were injected with equivalent volumes of saline. All mice were injected daily at 7 pm for the duration of the experiment. All experimental procedures were performed in accordance to the Institut Pasteur ethical committee and the French Ministry of Research (APAFiS; 16380).

All animal studies were performed as approved by the Harvard Committee on Animals. Adult (1-year-old) C57Bl/6 male mice were obtained from Charles River, and intravenously injected (by tail vein injection) with 0.5 mg/kg GDF11 (PeproTech) or 0.5 mg/kg, 1 mg/kg, 2 mg/kg and 4 mg/kg GDF8 (R&D Systems) or saline as control. Ligands were reconstituted in water at a concentration of 1 mg/mL and diluted in saline prior to injection. Heart tissue was collected 1 h post injection. Whole heart protein lysates were obtained by homogenizing the heart in RIPA buffer freshly supplemented with 1 mM PMSF and protein phosphatase inhibitor 2 and 3 (Sigma-Aldrich). 40 g total protein was loaded in NuPAGE 4-12% Bis-Tris gels (LifeTechnologies). Following transfer, membranes were blocked with non-fat dry milk for 1 h at room temperature and successively incubated with primary pSMAD2 antibody (Millipore, Cat. no. AB3849; Lot 2649232) and total SMAD2/3 antibody (Cell Signaling Technology, Cat. no. 8685P; Lot 4) overnight at 4 °C. Proteins were detected with horseradish peroxidase-conjugated antibodies (BioRad Laboratories; Cat. no. 172-1019; Lot L006328 A) and enhanced chemiluminescence (Amersham™ GE Healthcare, Cat. no. RPN2236).