BDNF is expressed in the adult heart and is secreted by cardiomyocytes. (A) Heart (H) and brain (B) lysates obtained from a knock-in BDNF HA tagged and a WT mouse used as a control were immunoprecipitated and blotted with anti-HA antibodies. 15-kD molecular mass marker. Lysate inputs are on the right of the panel. (B and C) Embryonic cardiomyocytes secrete biologically active BDNF. (B) Culture medium from WT and BDNF-HA cultured embryonic cardiomyocytes was immunoprecipitated as in A. WT and BDNF-HA brains were used as negative and positive controls, respectively. (C) Cells stably expressing TrkB tyrosine kinase were used as a sensitive biological assay to test for the presence of secreted biologically active BDNF in the medium of cultured cardiomyocytes. After a 10-min incubation with the medium from different cardiomyocyte cultures, cells expressing TrkB were lysed and subjected to Western analysis with an anti–phospho-TrkB antibody. Anti-TrkB and GAPDH antibodies were used as controls for loading. The tested supernatants were from WT, BDNF heterozygous (HET), and BDNF knockout cultured cardiomyocytes. Unconditioned media supplemented with 1 ng/ml of recombinant BDNF (BDNF)F and 1 µg/ml TrkB-Fc BDNF blocking antibody (BDNF+α) were used as positive and negative controls, respectively. Ctrl, unconditioned media (DMEM). The molecular mass is shown on the right.