Fig. 3
From: FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons
Yellow PfPKG biosensor monitors cGMP from GC-A and GC-B in cardiomyocytes. a Confocal images of a living cardiomyocyte expressing the Yellow PfPKG biosensor. Scale bar: 10 µm. b, c, e, f Recording of FRET ratio (CFP/Venus) in single cardiomyocytes expressing the Yellow PfPKG biosensor and stimulated with the NO donor SNAP (100 μM), the PDE inhibitor IBMX (100 µM), the sGC inhibitor ODQ (10 μM), BNP (300 nM) or CNP (300 nM), where indicated. FRET signal was normalized to that at the time of stimulation. Shown are traces representative of six cells from four animals (SNAP), eight cells from four animals (ODQ), five cells from four animals (BNP) or six cells from six animals (CNP). d Total cGMP levels in isolated ventricular cardiomyocytes in the absence (Ctr) or presence of 50 nM SNAP, 100 µM SNAP or 300 nM CNP (10 min stimulation). Data are mean ± SEM from five individual experiments. g Recording of FRET ratio (T-sapphire/Dimer2) in single cardiomyocytes expressing the Red cGES-DE5 biosensor and stimulated with CNP (300 nM) and IBMX (100 µM) (where indicated). FRET was normalized to that prior to stimulation. Shown are traces representative of four cells from four animals. h Recording of FRET ratio (CFP/Venus) in single cardiomyocytes expressing the Yellow PfPKG biosensor stimulated with increasing concentrations of CNP. Shown are traces representative of nine individual cells from five animals normalized to that prior to stimulation. i Concentration–response curves generated by stimulation with increasing concentrations of CNP in single cardiomyocytes expressing the indicated biosensor. Data are plotted as centiles and for the EC50 the mean ± SEM is indicated (nine cells from five animals for the Yellow PfPKG, six cells from six animals for the Red cGES-DE5). p = 0.37 (unpaired t-test). j Quantification of FRET responses shown in b, c, and e–g. p = 0.006 for CNP-stimulation for PfPKG vs. Red cGES-DE5 (one-way ANOVA with Sidak’s multiple comparisons test). k Maximal FRET response (CNP+IBMX) from cardiomyocytes expressing the indicated biosensor. Data are mean ± SEM from 34 cells from 27 animals (Yellow PfPKG) and 16 cells from 10 animals (Red cGES-DE5). *p = 0.0001 vs. Red cGES-DE5 (Two-tailed Student`s t test)
