Inhibition of adenylyl cyclase 1 by ST034307 inhibits IP3-evoked changes in sino-atrial node beat rate

Abstract

Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP3 signalling pathway has been proposed as an atrial-specific target for AF therapy, and atrial IP3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. We investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sino-atrial node (SAN) cells to the α-adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.5% to 8.2% (p = 0.005) in the presence of 1 μM ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.2%, ST034307 = 16.3%; p > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transients (CaT) generated by 10 μM PE in the presence and absence of 1 μM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; p = 0.003) but did not inhibit changes in CaT amplitude in response to PE in atrial cells. The results presented here demonstrate pharmacologically the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP3R calcium release.

Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP

“…These data support the involvement of AC1 stimulation following IP3R Ca 2+ release in regulation of SAN pacemaker activity (23, 37, 54, 56). As co-expression of AC1 and RyR2 in the SAN (56) appears to be comparable to that of AC8 and RyR2 in atrial cells (23), it is possible that a similar link between IP3R Ca 2+ release and activation of AC8 is present in atrial cardiomyocytes. Furthermore, overexpression of AC8 in mouse cardiac cells has been shown to result in increased contractility, independent of I Ca,L , demonstrating that endogenous AC8 may have the ability to influence cardiac contractility via a mechanism independent of AC5/6 (83).…”

“…Ca 2+ released via IP3R may directly lead to the activation of Ca 2+ -sensitive AC isoforms AC1 and/or AC8 in cardiac cells. Whilst such a link has not yet been shown directly in atrial cells, in the SAN selective inhibition of Ca 2+ -sensitive AC1 using ST034307 results in a decrease in spontaneous beating rate (56), whereas chelation of Ca 2+ using BAPTA and inhibition of ACs using MDL-12,330A reduces the hyperpolarisation activated ‘funny’ current (I f ) (70). These data support the involvement of AC1 stimulation following IP3R Ca 2+ release in regulation of SAN pacemaker activity (23, 37, 54, 56).…”

“…Whilst such a link has not yet been shown directly in atrial cells, in the SAN selective inhibition of Ca 2+ -sensitive AC1 using ST034307 results in a decrease in spontaneous beating rate (56), whereas chelation of Ca 2+ using BAPTA and inhibition of ACs using MDL-12,330A reduces the hyperpolarisation activated ‘funny’ current (I f ) (70). These data support the involvement of AC1 stimulation following IP3R Ca 2+ release in regulation of SAN pacemaker activity (23, 37, 54, 56). As co-expression of AC1 and RyR2 in the SAN (56) appears to be comparable to that of AC8 and RyR2 in atrial cells (23), it is possible that a similar link between IP3R Ca 2+ release and activation of AC8 is present in atrial cardiomyocytes.…”

“…-activated AC isoforms, AC1 or AC8, which are both expressed in close proximity to IP3R in atrial and SAN cells (23,(54)(55)(56), and are thought to influence SAN pacemaker activity through influence on the pacemaker current (I f ) (57). Increases in SAN beating rate in response to activation of α-AR by phenylephrine (PE) are dependent upon AC1 activity as shown using the AC1-selective inhibitor ST034307 (56), however it remains unclear whether Ca…”

“…A possible explanation for the involvement of AC activity downstream of IP3R Ca 2+ release is the direct stimulation of Ca 2+ -activated AC isoforms, AC1 or AC8, which are both expressed in close proximity to IP3R in atrial and SAN cells (23, 54–56), and are thought to influence SAN pacemaker activity through influence on the pacemaker current ( I f ) (57). Increases in SAN beating rate in response to activation of α-AR by phenylephrine (PE) are dependent upon AC1 activity as shown using the AC1-selective inhibitor ST034307 (56), however it remains unclear whether Ca 2+ -activated ACs are, directly or indirectly, involved in the downstream effects of IP3 signalling, or whether activation of IP3R leads to a Ca 2+ -dependent activation of AC and localised changes in cAMP within cardiomyocytes. Fluorescence resonance energy transfer (FRET)-based sensors enable real-time measurement of intracellular cAMP signalling and can be used to demonstrate compartmentalisation of cAMP/PKA signalling (14, 15, 58).…”