PDE Effects in Sinoatrial Node and Atrium Figure 5. Effects of the PDE4 inhibitor rolipram on action potential firing in SAN and atrial myocytes. A. Representative spontaneous AP recordings (5 s duration) in control conditions, in the presence of Rol (10 mM) and after Rol washout. Dotted lines are at 0 mV. Summary bar graphs illustrate the effects of Rol 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 Rol (10 mM) and after Rol washout. Dotted line is at 0 mV. F. Summary of the effects of Rol on atrial AP duration (APD50, APD70, and APD90). Summary data are means 6 SEM; n = 10 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.g005 atrial myocytes EHNA increased (P,0.05) ICa,L density from 23.460.2 to 24.960.5 pA/pF (Figure 7E). Furthermore, ICa,L Gmax in right atrial myocytes was increased (P,0.05) from 72.862.3 to 99.963.2 pS/pF by EHNA and the V1/2(act) was shifted (P,0.05) from 216.061.2 to 218.761.4 mV (Figure 7F). These data are consistent with the effects of EHNA on AP properties in SAN and atrial myocytes. In contrast to SAN and atrial myocytes, EHNA had no effect on right ventricular myocyte ICa,L (Figure S2). Summary I–V curves (Figure S2B) show no change (P = 0.92) in ICa,L density upon application of EHNA. Steady state conductance analysis (Figur- e S2C) shows that ICa,L Gmax (76.362.6 pS/pF in control vs. 76.162.9 pS/pF in EHNA) and V1/2(act) (21361 mV in control vs. 214.461.2) were not altered (P = 0.85) in the presence of EHNA. Thus, although selective PDE2 inhibition does not affect ventricular myocyte ICa,L, it does increase ICa,L in SAN and atrial myocytes. The effects of the PDE3 inhibitor Mil (10 mM) on SAN and right atrial ICa,L are illustrated in Figure 8. Consistent with its effects on spontaneous AP firing, Mil increased (P,0.05) peak ICa,L density in SAN myocytes from 23.860.5 to 26.360.2 pA/ pF (Figure 8B). Also, ICa,L Gmax was increased (P,0.05) from 110.765.8 to 160.463.3 pS/pF and V1/2(act) was shifted (P,0.05) from 216.862.3 to 218.462.1 mV by Mil (Figure 8C). In contrast to SAN myocytes, and in agreement with the absence of effects of PDE3 inhibition on right atrial AP duration, Mil had no effect on right atrial ICa,L. Summary I–V curves (Figure 8E) show no change (P = 0.66) in peak ICa,L density upon application of Mil. Furthermore, ICa,L Gmax (95.162.9 pS/pF in control vs. 100.962.3) and V1/2(act) (210.461.2 mV in control vs. 29.960.9 mV in Mil) were not altered following PDE3 inhibition in atrial myocytes. The effects of Mil on right ventricular myocyte ICa,L are illustrated in Figure S3. Summary I–V curves show that Mil had no effect (P = 0.63) on peak ICa,L density (Figure S3B). Also, ICa,L Gmax (87.261.8 pS/pF in control vs. 91.361.4 pS/pF in Mil) and V1/2(act) (28.860.6 mV in control vs. 29.260.4 mV in Mil) were not affected (P = 0.52) by Mil (Figure S3C). Together, these data show that PDE3 inhibition alone markedly increases ICa,L in the specialized pacemaker myocytes of the SAN, but has no effect on ICa,L in working atrial or ventricular myocytes. The effects of PDE4 inhibition with Rol (10 mM) on SAN and right atrial myocyte ICa,L are presented in Figure 9. In SAN myocytes Rol increased (P,0.05) peak ICa,L density from 24.260.1 to 28.161.1 pA/pF (Figure 9B). Rol also increased (P,0.05) ICa,L Gmax from 99.267.2 to 188.362.8 pS/pF and shifted (P,0.05) the V1/2(act) from 218.163.1 to 220.260.7 mV (Figure 9C). In right atrial myocytes Rol increased (P,0.05) peak ICa,L density from 24.560.4 to 27.560.7 pA/pF (Figure 9E). Furthermore, Rol increased (P,0.05) ICa,L Gmax from 130.860.7 PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e47652