(A and B) Neonatal cardiomyocytes expressing cAMP biosensor ICUE3 were stimulated with ISO in the absence and presence of pretreatment with insulin (100 nM, 30 min) or as indicated. The changes in cAMP FRET ratio were recorded. (A) Traces show time-courses of changes in FRET ratio in representative cells. (B) The maximal increases in FRET ratio were plotted. Neonatal cardiomyocytes expressing cAMP biosensor ICUE3 were stimulated with insulin, forskolin, or PDE inhibitor IBMX as indicated (C), or were pretreated with insulin together with either 1μM of ICI118551 or 1μM of CGP20712A before stimulation with ISO (D). The changes in cAMP FRET ratio were recorded. The maximal increases in FRET ratio were plotted (C and D). (C) ** p < 0.01, *** p < 0.001 by student t-test in relative to insulin (100nM) group (n = 6~8). (D) * p < 0.05 by one-way ANOVA followed by Tukey’s test (n = 12~14).