| dc.description.abstracteng | 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. | de |