PDE1 inhibition and adenosine A2A receptor activation synergistically potentiates spontaneous calcium waves and transients in mouse ventricular myocytes

Abstract

Background: The phosphodiesterase-1C (PDE1C) and the adenosine A2A receptor (A2AR) are located in a macromolecular cluster and expected to modulate cAMP-dependent regulation of the intracellular calcium homeostasis in cardiac myocytes. In this contect, atrial fibrillation has been associated with a high incidence of spontanoeus arrhythmogenic calcium waves caused by excessive A2AR activation. On the other hand, inhibition of PDE1C and A2AR activation has also been reported to produce a synergistic and protective effect on cardiomyocyte survival, but little is known about the corresponding effects on the intracellular calcium homeostasis.

Purpose: This study, therfore, aimed to investigate how PDE1 inhibition and/or A2AR activation modulate sarcoplasmic reticulum (SR) calcium homeostasis in mouse ventricular myocytes.

Methods: Mouse ventricular myocytes (47 cells from 20 mice) were loaded with the calcium indicator Rhod-2 and spontaneous calcium release events at rest or caffeine induced calcium transients were visualized using resonance-scanning confocal microscopy at a frame rate of 150 Hz. The SR calcium content was estimated as the ratio of the peak fluorescence and the fluoresence the end of a 10 mM caffeine pulse (20s). Calcium leak was estimated as ratio of the flourescence emmision at baseline before and at the end of the caffeine pulse.

Results: The incidence of spontaneous calcium waves or transients increased from 0.7±0.3 events/min in control to 1.1±0.5 events/min and 3.4±1.0 events/min (p<0.01) after inhibition of PDE1 with the selective antagonist ITI-214 and the A2AR agonist CGS-21680 respectively. Concomitant PDE1 inhibition and A2AR activation synergistically increased the event frequency to 8.4±2.3 events/min (p<0.05). There were no significant differences in the corresponding SR calcium contents (4.4±0.3, 4.5±0.4, 4.7±0.4 and 4.1±0.2 for control, ITI-214, CGS-21680 and ITI-214+CGS-21680 respectively). However, there was a parallel increase in calcium leak from 8.7±4.2% in control to 21.8±9.8% with ITI-214, 17.6±7.2% with CGS-21680 and 37.5±11.1% when myocytes were exposed to both compounds (p<0.05), suggesting that the higher incidence of spontaneous calcium waves and transients observed upon PDE1 inhibition and/or A2AR activation is likely caused by a lowering of the threshold for spontaneous SR calcium release rather than by SR calcium overloading.

Conclusion: PDE1 inhibition exacerbates the incidence of spontaneous arrhythmogenic SR calcium release events in myocytes with high levels of A2AR activation, suggesting that PDE1 inhibition under these conditions increases the risk of arrhythmia.