PDE Effects in Sinoatrial Node and Atrium Figure 6. Effects of IBMX on L-type Ca2+ current in SAN and right atrial myocytes. A. Representative ICa,L recordings in SAN myocytes in control conditions, in the presence of IBMX (100 mM) and following IBMX washout. B. Summary I–V relationships for the effects of IBMX on SAN myocyte ICa,L. C. Summary ICa,L conductance density plots for the effects of IBMX in SAN myocytes. D. Representative ICa,L recordings in right atrial myocytes in control conditions, in the presence of IBMX (100 mM), and following IBMX washout. E. Summary I–V relationships for the effects of IBMX on right atrial myocyte ICa,L. F. Summary ICa,L conductance density plots for the effects of IBMX in right atrial myocytes. Summary data are means 6 SEM; n = 8 SAN myocytes and 12 right atrial myocytes; *P,0.05 vs. control by paired Student’s t-test. doi:10.1371/journal.pone.0047652.g006 to 176.961.0 pS/pF and shifted (P,0.05) the V1/2(act) from 27.160.2 to 212.360.2 mV (Figure 9F). These effects of PDE4 inhibition on ICa,L are consistent with the ability of Rol to augment spontaneous AP firing in SAN myocytes and AP duration in atrial myocytes. In contrast to SAN and atrial myocytes, Rol had no effect on right ventricular myocyte ICa,L (Figure S4). Summary I–V curves illustrate no change (P = 0.13) in peak ICa,L density following application of Rol. In addition, neither ICa,L Gmax (98.561.5 pS/ pF in control vs. 95.161.9 pS/pF in Rol) or V1/2(act) (29.160.4 mV in control vs. 28.960.6) were modulated (P = 0.53) by Rol. Thus, selective PDE4 inhibition elicits increases in ICa,L in SAN and atrial myocytes, but not in ventricular myocytes. The observation that selective PDE inhibitors, when given alone, have no effect on basal ICa,L in ventricular myocytes is consistent with previous studies in mice and rats, which have shown that selective inhibitors must be given in combination to augment basal ICa,L [10,11]. We have confirmed this pattern in our experiments by demonstrating that combined inhibition of PDE3 and PDE4 with Mil and Rol increases right ventricular myocyte ICa,L (Figure S5). Summary I–V curves illustrate that Mil + Rol increased peak ICa,L density (24.560.3 pA/pF in control vs. 26.760.6 pA/pF in Mil + Rol; P,0.05), increased ICa,L Gmax (84.861.5 pS/pF in control vs. 105.563.9 pS/pF in Mil + Rol; P,0.05) and shifted the V1/2(act) (211.260.5 mV in control vs. 220.861.1 mV in Mil + Rol; P,0.05) compared to control. Thus, our data illustrating the effects of global and specific PDE inhibitors on ventricular ICa,L fully agree with previous studies, which supports the conclusion that specific PDE inhibitors have distinct effects in different regions of the myocardium. These ventricular ICa,L data also confirm that the PDE inhibitors we have used are selective at the doses we have studied. We also measured the effects of combined PDE3/4 inhibition on AP firing and ICa,L in SAN and right atrial myocytes. We observed that Mil + Rol augments AP firing and ICa,L in SAN and atrial myocytes similarly to IBMX (data not shown). Discussion PDEs are critical regulators of cAMP and cGMP levels in cardiomyocytes, representing the primary enzymes responsible for cyclic nucleotide degradation, and acting as the only counter balance to the mechanisms responsible for cyclic nucleotide production (adenylyl and guanylyl cyclases) [1,2]. In this study, we have measured the effects of global (IBMX) and specific (EHNA, Mil, Rol) PDE inhibitors on AP firing and ICa,L in mouse SAN and atrial myocytes. We also measured the effects of these inhibitors on mouse ventricular ICa,L in order to make direct comparisons between the three myocyte types. This is the first direct comparison of these effects in the mouse myocardium. The main finding of these experiments is that PDE effects on AP firing and basal ICa,L are distinct in the different regions of the myocardium. Our AP data clearly demonstrate that global PDE inhibition with IBMX potently increases spontaneous AP frequency in mouse SAN myocytes in association with increases in DD slope and APD50. Similarly IBMX markedly increased AP duration at 50, 70 and 90% repolarization in mouse atrial myocytes. These data show that there is a high level of constitutive PDE activity that regulates AP properties in the SAN and atrial myocardium; however, subsequent experiments with specific PDE inhibitors PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e47652