Real time visualization of cGMP and cAMP dynamics in intact adult cardiomyocytes using new transgenic mice

cAMP and cGMP are important second messengers in the cardiovascular system. Previously, it has been demonstrated that the cardiac ryanodine receptor (RyR2) forms a microdomain associated with key contributors of cAMP signaling such as PKA, PDE4D3 and various phosphatases. Its calcium release channel function is highly regulated by cAMP and its effector kinase PKA; hyperphosphorylation of the channel occurs in heart failure and causes arrhythmias. A transgenic mouse model was generated with cardiomyocyte specific expression of the FRET-based cAMP indicator Epac1-camps-JNC. Epac1-camps-JNC is a targeted version of the cytosolic cAMP sensor Epac1-camps by its fusion with junctin protein, which forms a complex with calcium release units to permit locally restricted real time visualization of cAMP dynamics in vicinity of the RyR2. Stimulation of the β1-adrenergic receptor strongly increased cAMP levels, while the β2-induced signals were hardly detectable in this microdomain. Furthermore, it was found that PDE3 and PDE4 are the major contributors to local cAMP catabolism. cAMP dynamics recorded with the localized Epac1- camps-JNC were compared with cAMP signaling in the bulk cytosol using cardiomyocytes isolated from Epac1-camps mice. Yet there were no significant differences detectable in cAMP signaling between the cytosol and the RyR2 compartment. cGMP is another important second messenger which is considered cardioprotective, and the agents that raise cGMP are under investigation in clinical trials for hypertension and heart failure. However, little is known about the spatio-temporal dynamics of cGMP in adult cardiomyocytes. Therefore, a new transgenic mouse model with cardiomyocyte-specific expression of the FRET-based biosensor red cGES-DE5 was generated to allow real time visualization of cGMP dynamics with nanomolar sensitivity in living adult cardiomyocytes. In these cells, basal cGMP levels were amounted to ~10 nmol/L. It was found that they are generated by NO-GC activity and β3-adrenergic receptors are involved in the regulation of unstimulated cGMP levels. A strong increase upon stimulation of GC-B with CNP was observed, while ANP which stimulates GC-A evoked only minor effects. In contrast, NO donors such as SNAP failed to raise cGMP via activation of NO-GC, most likely due to low expression levels of NO-GC in adult cardiomyocytes. Surprisingly, cGMP degradation was mainly mediated by PDE3 hydrolytic activity, while PDE1, 2 and 5 seemed to be not involved in cGMP turnover. In a model of compensated cardiac hypertrophy, PDE3 remained the major cGMP-PDE and PDE5 activity was upregulated. PDE3 is well known as a major cAMP-PDE and regulator of myocyte contractility. However, it is also established that PDE3 is inhibited by cGMP, since it degrades both cyclic nucleotides with high affinity but has a 10- fold higher catalytic activity for cGMP compared to cAMP. To elucidate the role of PDE3 in cyclic nucleotide interplay, cardiomyocytes isolated from transgenic mice expressing the cytosolic cAMP sensor Epac1-camps were subjected to FRET-based measurements of cAMP. After β-adrenergic stimulation, application of CNP increased cAMP levels due to cGMP-mediated PDE3 inhibition, thus confirming the major role of PDE3 in cGMP/cAMP crosstalk.