BDNF signals independently of the catecholaminergic pathway and does not increase cardiac CamKII or PLB phosphorylation at the peak of its inotropic function. (A) Isoproterenol does not occlude the effect of BDNF in Langendorff-perfused hearts. Representative LVDP trace obtained from application of 100 nM isoproterenol (ISO) to a Langendorff-perfused heart. Once the heart reached stable LVDP a 50 µl (10 ng total) bolus of BDNF was applied followed by a second 50 µl (5 mM) bolus of caffeine as a positive control 2 min later. (B) Quantification of the effect of BDNF and caffeine on baseline LVDP in the absence or presence of ISO. The baseline for the values obtained with ISO is considered LVDP in the presence of ISO. Values are from four independent experiments. (C–F) BDNF does not increase cardiac CamKII or PLB phosphorylation at the peak of its inotropic function. (C) Western blot analysis of PLB phosphorylation at Ser16 (PLB P-S16) and Thr17 (PLB P-T17), CAMKII phosphorylation at Thr 286/287 (CAMKII P-S286), and Troponin I at Ser23/24. Langendorff-perfused hearts were injected with vehicle (negative control), BDNF, or Forskolin (positive control) as described in Material and methods. (D–F) Quantification of the bands intensity (±SEM) from C reported as the ratio between the phosphorylated form over the specific total protein. Means of the three samples ±SEM are shown. *, P < 0.05, calculated with two-tail t test.