PDE Effects in Sinoatrial Node and Atrium Figure 3. Effects of the PDE2 inhibitor EHNA on action potential firing in SAN and atrial myocytes. A. Representative spontaneous AP recordings (5 s duration) in control conditions, in the presence of EHNA (10 mM) and after EHNA washout. Dotted lines are at 0 mV. Summary bar graphs illustrate the effects of EHNA on spontaneous AP frequency (B), DD slope (C) and APD50 (D). E. Representative stimulated right atrial myocyte APs in control conditions, in the presence of EHNA (10 mM) and after EHNA washout. Dotted line is at 0 mV. F. Summary of the effects of EHNA on atrial AP duration (APD50, APD70, and APD90). Summary data are means 6 SEM; n = 6 SAN myocytes and 7 right atrial myocytes; *P,0.05 vs. control by one way ANOVA with Tukey’s posthoc test. doi:10.1371/journal.pone.0047652.g003 atrial myocytes are not known. Thus, we next studied the effects of the specific PDE inhibitors erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA; PDE2 inhibitor; 10 mM) [25], milrinone (Mil; PDE3 inhibitor; 10 mM) [10,20] and rolipram (Rol; PDE4 inhibitor; 10 mM) [10]. The selectivity of these inhibitors for specific PDE families is well established, but also concentration dependent. Accordingly, we have used each of these compounds at concentrations that have been previously shown to be selective to their respective PDE family [3,10,11,26,27]. The effects of PDE2 inhibition with EHNA on AP firing in mouse SAN and right atrial myocytes are presented in Figure 3 (see also Tables S3 and S4). EHNA increased (P,0.05) sponta- neous AP frequency in SAN myocytes from 13767 to 16066 APs/min (Figure 3B). In addition, EHNA increased (P,0.05) DD slope (28.961.4 mV/s in control vs. 41.2 mV/s in EHNA; Figure 3C) and APD50 (33.663.8 ms in control vs. 39.664.1 ms in EHNA; Figure 3D). EHNA also increased (P,0.05) AP duration in mouse right atrial myocytes (Figure 3F). Specifically, APD50 was increased from 9.461.1 to 1261.1 ms, APD70 was increased from 17.562.3 to 22.362.3 ms and APD90 was increased from 4564.6 to 50.565.2 ms. The effects of EHNA were reversed upon washout. These data demonstrate that PDE2 constitutively regulates AP firing in mouse SAN and atrial myocytes in basal conditions. The effects of the PDE3 inhibitor Mil on AP firing in mouse SAN and right atrial myocytes are illustrated in Figure 4 (see also Tables S5 and S6). Mil increased (P,0.05) spontaneous SAN myocyte AP frequency from 13566 to 16966 APs/min (Figure 4B). Mil also increased (P,0.05) DD slope from 28.563.4 to 49.964.5 mV/s (Figure 4C) and APD50 from 3765.4 to 46.265.5 ms (Figure 4D) in SAN myocytes. These effects of Mil on SAN myocytes were fully reversible. In contrast to the SAN, Mil had no effect on AP properties in mouse right atrial myocytes. APD50 (8.961 vs. 9.461), APD70 (16.962 vs. 18.262.3) and APD90 (45.764.2 vs. 4664.3) were not different (P = 0.79; Figure 4F) upon application of Mil. These data indicate that the mouse SAN has constitutive PDE3 activity that regulates spontaneous AP firing but that PDE3 inhibition does not modulate atrial AP properties in basal conditions. The effects of the PDE4 inhibitor Rol on AP firing in mouse SAN and right atrial myocytes were measured next (Figure 5, Tables S7 and S8). Rol increased (P,0.05) spontaneous AP frequency in SAN myocytes from 13068 to 17069 APs/min (Figure 5B). In addition, Rol increased (P,0.05) DD slope (23.961.8 mV/s vs. 44.364.3 mV/s; Figure 5C) and APD50 (37.565 ms vs. 47.866.3 ms; Figure 5D) in SAN myocytes. Rol also increased (P,0.05) AP duration in right atrial myocytes (Figure 5F). Specifically, APD50 was increased from 9.461.7 to 12.962 ms, APD70 was increased from 19.963.4 to 27.463.9 ms, and APD90 was increased from 51.165.9 to 59.165.5 ms. These data show that constitutive PDE4 activity contributes to the regulation of AP firing in mouse SAN and atrial myocytes. PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47652