The total enzymatic PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724. The protein expression of PDE4A, 4B and 4D was evaluated by Western blot using specific antibodies for each variant. The in vitro pacemaker activity was assessed by measuring the spontaneous Ca2+ transients in Fluo4-loaded-SAN intact tissue. Images were obtained using confocal microscopy.
Inhibition of PDE4 by Ro-20-1724 increased the beating rate of intact mouse SAN. PDE4 inhibition also altered Ca2+ homeostasis, as shown by the presence of irregular Ca2+ transients and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban) after treatment with Ro-20-1724. PDE4 enzymatic activity was found to account for 60% of the total cAMP-PDE activity in SAN. The 3 isoforms PDE4A, 4B and 4D were found to be expressed in mouse SAN.
Our preliminary results reveal the involvement of PDE4 in the control of pacemaker activity in SAN. The characterization of pacemaker activity in PDE4B and PDE4D-null mice is under investigation.
The author hereby declares no conflict of interest