PDE1 inhibition potentiates arrhythmogenic disturbances in calcium homeostasis and electrical activity induced by pathological adenosine levels in mouse ventricular myocytes

Abstract Background Phosphodiesterase-1 (PDE1) and adenosine A2A receptors (A2AR) are located in a macromolecular cluster and expected to modulate cAMP-dependent modulation of the intracellular calcium homeostasis in cardiac myocytes. However, the natural ligand adenosine (ADO) also activates adenosine A1 and A3 receptors that may have opposite effect on cAMP-signaling. Purpose This study aimed to investigate how PDE1 and adenosine A2AR-inhibition affects intracellular calcium homeostasis and arrhythmogenesis upon an increase in extracellular adenosine levels that mimic pathological elevations of adenosine observed during ischemia and in plasma from patients with atrial fibrillation. Methods Mouse ventricular myocytes were loaded with the calcium indicator Rhod-2. Spontaneous calcium release and calcium transients, elicited by electrical field stimulation, were visualized using resonance-scanning confocal microscopy in myocytes from 7 mice. Perforated patch-camp technique was used to measure spontaneous membrane depolarizations in myocytes from 7 mice. Results Adenosine (10 µM) increased the incidence of spontaneous calcium waves or transients from 0.11±0.11 in control to 0.56±0.17 events/min (p0.05). Subsequent inhibition of PDE1 with 1 µM of the selective antagonist ITI214 (ITI) dramatically increased the incidence of calcium waves to 4.1±1.3 events/min (p0.01). Similarly, ADO+ITI significantly increased the incidence of arrhythmic responses in myocytes paced at 1 Hz from 0/9 cells in control to 8/9 cells (p0.05), and preincubation with 200 nM of the A2AR antagonist ZM241385 prevented the induction of arrhythmic responses. ADO and ADO+ITI treatments had no significant impact on RyR2 phosphorylation at S2814. However, ADO and ADO+ITI strongly increased RyR2 phosphorylation at S2808 (2- and 2.2-fold respectively) and ZM241385 prevented the increases in S2808 phosphorylation induced by ADO+ITI. Interestingly, ADO+ITI had no effect on the amplitude or incidence of small membrane depolarizations (less than 10 mV) in resting myocytes or when paced at 1 Hz. However, ADO+ITI induced long-lasting (19.1±4.2s) spontaneous membrane depolarizations with a plateau-potential of -18.6±16.5 mV in 6/10 myocytes at rest. These depolarizations were also observed in 5/10 myocytes when paced at 1 Hz with an average duration of 20.6±4.7s, and a plateau-potential of -17.9±20.2 mV. Importantly, subsequent exposure to ZM241385 abolished the long-lasting membrane depolarizations in all myocytes. Conclusion PDE1 inhibition exacerbates the incidence of spontaneous arrhythmogenic SR calcium release events, afterdepolarizations and arrhythmic beating patterns elicited by pathological levels of extracellular adenosine, which can be reversed by selective A2AR inhibition. This points to the A2ARs as a target for preventing arrhythmia induced by pathological elevations of extracellular adenosine levels.