Activation of IPR in atrial cardiomyocytes leads to generation of cytosolic cAMP.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the inositol (1,4,5)-trisphosphate (IP) signaling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET)-based cytosolic cyclic adenosine monophosphate (cAMP) sensor EPAC-S in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, the addition of the α-agonist, phenylephrine (PE, 3 µM), resulted in a FRET change of 21.20 ± 7.43%, and the addition of membrane-permeant IP derivative 2,3,6-tri---myo-IP(1,4,5)-hexakis(acetoxymethyl)ester (IP-AM, 20 μM) resulted in a peak of 20.31 ± 6.74%. These FRET changes imply an increase in cAMP. Prior application of IP receptor (IPR) inhibitors 2-aminoethyl diphenylborinate (2-APB, 2.5 μM) or Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to IP-AM. The FRET change in response to PE in NRVMs was not inhibited by 2-APB or Xestospongin-C. Finally, the localization of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin-C. These data support further investigation of the proarrhythmic nature and components of IP-induced cAMP signaling to identify potential pharmacological targets. This study shows that indirect activation of the IP pathway in atrial myocytes using phenylephrine and direct activation using IP-AM leads to an increase in cAMP and is in part localized to the cell membrane. These changes can be pharmacologically inhibited using IPR inhibitors. However, the cAMP rise in ventricular myocytes is independent of IPR calcium release. Our data support further investigation into the proarrhythmic nature of IP-induced cAMP signaling.