PDE Effects in Sinoatrial Node and Atrium Figure 2. Effects of IBMX on action potential firing in SAN and atrial myocytes. A. Representative spontaneous AP recordings (5 s duration) in control conditions, in the presence of IBMX (100 mM) and after IBMX washout. Dotted lines are at 0 mV. Summary bar graphs illustrate the effects of IBMX 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 IBMX (100 mM) and after IBMX washout. Dotted line is at 0 mV. F. Summary of the effects of IBMX on atrial AP duration (APD50, APD70, and APD90). Summary data are means 6 SEM; n = 11 SAN myocytes and 13 right atrial myocytes; *P,0.05 vs. control by one way ANOVA with Tukey’s posthoc test. doi:10.1371/journal.pone.0047652.g002 myocardium; however, have not been determined. Accordingly, we have measured the mRNA expression of PDE2A, PDE3A, PDE3B, PDE4A, PDE4B and PDE4D in the SAN, right atrium and right ventricular free wall using quantitative PCR (Figure 1A). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. These three regions of myocardium coincide with the cell types used in our electrophysiology experiments (see below). SAN samples were distinguished from right atrial samples based on the high expression of HCN4 and low expression of ANP in the SAN compared to the atrium (Figure 1B) as previously described by us [20] and others [24]. These data demonstrate that each region of the myocardium has a specific pattern of expression for the PDE2, 3 and 4 isoforms we have measured. Specifically, no differences were observed in the expression of PDE2A in each region of the myocardium; however, PDE3A, PDE3B, PDE4A, PDE4B and PDE4D all displayed significant differences in expression between SAN, right atrium and right ventricle as indicated in Figure 1A. Effects of global PDE inhibition on SAN and right atrial myocyte APs IBMX is a broad spectrum PDE inhibitor that antagonizes all PDE isoforms [1,11]. In our initial electrophysiological studies we measured the effects of IBMX (100 mM) on spontaneous AP firing in SAN myocytes and stimulated APs in right atrial myocytes (Figure 2; Tables S1 and S2). IBMX increased (P,0.05) spontaneous AP firing in SAN myocytes from 138610 to 193614 APs/min with no change in maximum diastolic potential (MDP; Figure 2B, Table S1). This change in AP frequency was associated with increases (P,0.05) in DD slope (28.364 mV/s in control vs. 58.4 4.1 mV/s in IBMX; Figure 2C) and APD50 (35.364.5 ms in control vs. 55.165.3 ms in IBMX; Figure 2D). Additional SAN AP parameters are shown Table S1, which also shows that the effects of IBMX on SAN myocyte AP firing are fully reversible upon washout. In right atrial myocytes, IBMX increased (P,0.05) AP duration at 50, 70 and 90% repolarization (APD50, APD70, APD90) with no change in resting membrane potential (RMP; Figure 2F, Ta- ble S2). Specifically, APD50 was increased from 8.260.9 to 16.962.1 ms, APD70 was increased from 12.963.7 to 32.264.3 ms and APD90 was increased from 47.764.5 to 60.266.6 ms. Additional atrial AP parameters are shown in Table S2. The effects of IBMX on right atrial APs were reversed upon drug washout. Effects of selective PDE2, PDE3 and PDE4 inhibitors on SAN and right atrial myocyte APs PDE2, PDE3 and PDE4 have been shown to be major contributors to total PDE activity in rodent ventricular myocytes [10,11]; however, their relative contributions in mouse SAN and PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e47652