PDE Effects in Sinoatrial Node and Atrium Figure 9. Effects of the PDE4 inhibitor rolipram 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 Rol (10 mM) and following Rol washout. B. Summary I–V relationships for the effects of Rol on SAN myocyte ICa,L. C. Summary ICa,L conductance density plots for the effects of Rol in SAN myocytes. D. Representative ICa,L recordings in right atrial myocytes in control conditions, in the presence of Rol (10 mM), and following Rol washout. E. Summary I–V relationships for the effects of Rol on right atrial myocyte ICa,L. F. Summary ICa,L conductance density plots for the effects of Rol in right atrial myocytes. Summary data are means 6 SEM; n = 6 SAN myocytes and 12 right atrial myocytes; *P,0.05 vs. control by paired Student’s t-test. doi:10.1371/journal.pone.0047652.g009 SR Ca2+ release mechanism is also cAMP sensitive and has been shown to be constitutively regulated by PDE3 [34]. Other possible targets include cyclic nucleotide sensitive delayed rectifier K+ currents, such as IKs, which can be modulated by IBMX and Mil in guinea pig SAN myocytes [41]. It is not known if mouse SAN expresses IKs, but it has been shown to express IKr channels that are thought to be cAMP sensitive [42]. Further studies will be needed to assess the possible contributions of each of these ionic currents to the changes in AP properties elicited by PDE inhibitors in mouse SAN. Interestingly, spontaneous AP firing in the SAN is also partially determined by a T-type Ca2+ current (ICa,T) [15,43,44]. Numerous studies have shown that ICa,T, including in SAN myocytes, is not modulated by cAMP [43,45–48]; thus, it is unlikely that ICa,T is affected by PDE inhibitors. It is also possible that other PDE families contribute to AP and ionic current regulation in SAN and atrial myocytes. For example, PDE1 and PDE5 have been shown to be expressed in the heart and to play functional roles in some settings [1,7,49,50]. More recently PDE8 has also been identified in ventricular myocardium [7]. Whether these PDE families have distinct roles in SAN or atrial myocytes in mice is yet to be determined. In summary, we have characterized the effects of inhibitors of PDE2, PDE3 and PDE4 on AP properties in mouse SAN and atrial myocytes and directly compared the effects of these inhibitors on basal ICa,L in SAN, right atrial and right ventricular myocytes. Although IBMX potently increased ICa,L in all cell types our data demonstrate that each region of the myocardium displays a unique pattern of response to family specific PDE inhibitors. SAN myocytes were responsive to each specific inhibitor tested, while atrial myocytes were responsive to PDE2 and PDE4, but not PDE3 inhibitors. Ventricular myocytes, on the other hand, do not respond to any family specific PDE inhibitors when given alone. These differences may be important when considering cyclic nucleotide regulation of ion channels in different regions of the heart. It is now known that PDE signaling in the heart is complex, based on the hypothesis that PDEs can be compartmentalized to distinct subcellular regions. Our data show that another aspect of the complexity of PDE regulation in the heart is related to regional differences in the roles of specific PDE families on ion channel regulation in the SAN compared to the working (atrial and ventricular) myocardium. Supporting Information Figure S1 Effects of IBMX on L-type Ca2+ current in right ventricular myocytes. A. Representative ICa,L record- ings (at 0 mV from 240 mV) in right ventricular myocytes in control conditions, in the presence of IBMX (100 mM), and after IBMX washout. B. Summary I–V relationships for the effects of IBMX on right ventricular ICa,L. C. Summary ICa,L conductance density plots for the effects of IBMX on right ventricular myocytes. Summary data are means 6 SEM; n = 6 ventricular myocytes; *P,0.05 vs. control by paired Student’s t-test. (TIF) Figure S2 Effects of PDE2 inhibition with EHNA on L- type Ca2+ current in right ventricular myocytes. A. Representative ICa,L recordings (at 0 mV from 240 mV) in right ventricular myocytes in control conditions, in the presence of EHNA (10 mM), and after EHNA washout. B. Summary I–V relationships for the effects of EHNA on right ventricular ICa,L. C. PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e47652