PDE Effects in Sinoatrial Node and Atrium Figure 4. Effects of the PDE3 inhibitor milrinone on action potential firing in SAN and atrial myocytes. A. Representative spontaneous AP recordings (5 s duration) in control conditions, in the presence of Mil (10 mM) and after Mil washout. Dotted lines are at 0 mV. Summary bar graphs illustrate the effects of Mil 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 Mil (10 mM) and after Mil washout. Dotted line is at 0 mV. F. Summary of the effects of Mil on atrial AP duration (APD50, APD70, and APD90). Summary data are means 6 SEM; n = 9 SAN myocytes and 9 right atrial myocytes; *P,0.05 vs. control by one way ANOVA with Tukey’s posthoc test. doi:10.1371/journal.pone.0047652.g004 Effects of PDE inhibition on L-type Ca2+ current in SAN and right atrial myocytes Numerous studies have demonstrated that ICa,L is a major target of regulation by PDEs, including PDE2, 3 and 4 [10,11,25,27,28] Furthermore, ICa,L is a critical determinant of spontaneous AP firing in the SAN [15–17] as well as AP duration in working atrial and ventricular myocytes [18,29,30]. Thus, we next studied the effects of PDE inhibitors on ICa,L in mouse SAN and right atrial myocytes. For comparison purposes, we also measured ICa,L in right ventricular myocytes. Importantly, some differences exist in SAN, atrial and ventricular ICa,L. Specifically, ICa,L in SAN and atrial myocytes is generated by both CaV1.2 and CaV1.3 channel isoforms [16,17]. This causes total ICa,L to activate between 260 and 250 mV and ICa,L I–V curves to peak between 210 and 0 mV. In contrast, ventricular ICa,L is determined only by CaV1.2 channels, activates positive to 240 mV and peaks at approxi- mately +10 mV. The effects of global PDE inhibition with IBMX (100 mM) on SAN and right atrial myocyte ICa,L are illustrated in Figure 6. Summary I–V curves demonstrate that IBMX increased (P,0.05) peak ICa,L density from 24.460.3 to 211.261 (Figure 6B) in SAN myocytes. The effects of IBMX on SAN myocyte ICa,L were further studied using steady state conductance analysis (Figure 6C). IBMX increased (P,0.05) ICa,L maximum conductance (Gmax) from 103.966.4 to 225.165.9 pS/pF and shifted (P,0.05) the V1/ 2(act) from 228.460.4 to 233.360.5 mV. There was no change (P = 0.588) in slope factor (6.460.6 mV in control vs. 6.360.3 mV in IBMX). Similarly, in right atrial myocytes, IBMX increased peak ICa,L density from 23.960.2 to 29.760.4 pA/pF (Figure 6E). Steady state conductance analysis in atrial myocytes (Figure 6F) shows that IBMX increased (P,0.05) ICa,L Gmax from 112.868.8 to 195.8610.4 pS/pF and shifted (P,0.05) the V1/2(act) from 29.161.2 to 218.761.1 mV. The effects of IBMX (100 mM) on ICa,L in right ventricular myocytes are illustrated in Figure S1. Summary I–V curves show that IBMX increased (P,0.05) peak ICa,L density from 24.960.7 to 211.260.9 pA/pF (Figure S1B) in ventricular myocytes. Furthermore, ICa,L Gmax in ventricular myocytes was increased (P,0.05) from 115615.2 to 197.1623 pS/pF and V1/2(act) was shifted (P,0.05) from 28.960.8 to 215.461 mV (Figure S1C) in IBMX. Thus, global PDE inhibition potently, and similarly, increases basal ICa,L in all regions of the myocardium we have investigated. Next we measured the effects of the PDE2 inhibitor EHNA (10 mM) on SAN and right atrial myocyte ICa,L (Figure 7). In SAN myocytes EHNA increased (P,0.05) peak ICa,L density from 24.760.4 to 26.160.5 pA/pF (Figure 7B). Steady state conductance analysis illustrates that EHNA increased (P,0.05) ICa,L Gmax from 106.067.7 to 125.565.2 pS/pF and shifted the V1/2(act) from 225.161.5 to 228.561.6 mV (Figure 7C). In right PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e47652