PDE Effects in Sinoatrial Node and Atrium Figure 7. Effects of the PDE2 inhibitor EHNA 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 EHNA (10 mM) and following EHNA washout. B. Summary I–V relationships for the effects of EHNA on SAN myocyte ICa,L. C. Summary ICa,L conductance density plots for the effects of EHNA in SAN myocytes. D. Representative ICa,L recordings in right atrial myocytes in control conditions, in the presence of EHNA (10 mM), and following EHNA washout. E. Summary I–V relationships for the effects of EHNA on right atrial myocyte ICa,L. F. Summary ICa,L conductance density plots for the effects of EHNA in right atrial myocytes. Summary data are means 6 SEM; n = 6 SAN myocytes and 10 right atrial myocytes; *P,0.05 vs. control by paired Student’s t-test. doi:10.1371/journal.pone.0047652.g007 revealed important differences between the two cell types. Specifically, in mouse SAN myocytes, EHNA, Mil and Rol each increased spontaneous AP frequency on their own, indicating that PDE2, PDE3 and PDE4 all contribute to the regulation of AP properties in basal conditions. In contrast, in mouse atrial myocytes, only EHNA and Rol were able to increase AP duration whereas Mil had no effect, indicating that constitutive PDE2 and PDE4, but not PDE3 activity, regulates atrial AP properties. Similar trends were also observed for ICa,L in SAN and atrial myocytes. Specifically, IBMX increased basal ICa,L by approxi- mately 185% in SAN myocytes and 140% in atrial myocytes. Consistent with our AP measurements, SAN myocyte ICa,L was increased by each of the selective inhibitors EHNA (,31%), Mil (,66%) and Rol (,93%). In atrial myocytes only EHNA (,38%) and Rol (,72%) increased basal ICa,L. Mil had no significant effect on right atrial ICa,L once again confirming a key difference between SAN and atrial myocytes in terms of which specific PDE inhibitors are able to modulate basal AP properties and ICa,L in mice. For further comparison, we measured the effects of IBMX on basal ICa,L in mouse right ventricular myocytes and observed increases of approximately 130%. In agreement with prior studies [8,10,11], but in contrast to our findings in SAN and atrial myocytes, none of the selective PDE inhibitors we tested had any effect on basal ICa,L in ventricular myocytes. Only when Mil and Rol were applied together was ventricular ICa,L increased as also noted previously [11]. Thus, although IBMX potently and similarly increases ICa,L in all regions of the myocardium, the ability of selective PDE inhibitors (EHNA, Mil or Rol) to modulate basal ICa,L is strikingly different in SAN, atrial and ventricular myocytes. We also measured the mRNA expression of specific isoforms from the PDE2, PDE3 and PDE4 family in SAN, right atrial and right ventricular myocardium using quantitative PCR. These data show that PDE2A, PDE3A, PDE3B, PDE4A, PDE4B and PDE4D are all expressed in each region of the myocardium and that several differences exist in expression levels between SAN, right atrium and right ventricular free wall. Note that these specific isoforms were chosen based on prior studies that have shown these same isoforms are detectable in atrial and ventricular myocardium or isolated ventricular myocytes [7,11,31]. Comparing our electrophysiological data with our mRNA expression data suggests the differences in ability of specific PDE inhibitors to modulate AP firing and basal ICa,L in the different regions of the myocardium are not strictly related to differences in expression. For example, PDE2A is similarly expressed in each region of the myocardium yet PDE2 inhibition with EHNA augments ICa,L in SAN and atrial myocytes, but not ventricular myocytes. Also, PDE3A (thought to be the main PDE3 isoform in cardiac myocytes [32,33]) is lowest in the SAN and highest in the ventricular myocardium; however, PDE3 inhibition with Mil only modulates basal ICa,L in SAN myocytes. Thus, it is likely that factors other than expression levels contribute importantly to the different effects of selective PDE inhibitors in different regions of the heart. These could include differences in subcellular localization of PDEs by anchoring proteins, differences in basal adenylyl and guanylyl cyclase activities, and differences in activity of PDE regulatory proteins in SAN, atrial and ventricular myocytes. It should also be noted that mRNA levels do not necessarily correlate with protein levels PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e47652