Summary ICa,L conductance density plots for the effects of EHNA on right ventricular myocytes. Summary data are means 6 SEM; n = 10 ventricular myocytes; EHNA had no effect on right ventricular ICa,L (paired Student’s t-test). (TIF) Figure S3 Effects of PDE3 inhibition with milrinone 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 Mil (10 mM), and after Mil washout. B. Summary I–V relationships for the effects of Mil on right ventricular ICa,L. C. Summary ICa,L conductance density plots for the effects of Mil on right ventricular myocytes. Summary data are means 6 SEM; n = 5 ventricular myocytes; Mil had no effect on right ventricular ICa,L (paired Student’s t-test). (TIF) Figure S4 Effects of PDE4 inhibition with rolipram 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 Rol (10 mM), and after Rol washout. B. Summary I–V relationships for the effects of Rol on right ventricular ICa,L. C. Summary ICa,L conductance density plots for the effects of Rol on right ventricular myocytes. Summary data are means 6 SEM; n = 5 ventricular myocytes; Rol had no effect on right ventricular ICa,L (paired Student’s t-test). (TIF) Figure S5 Effects of PDE3 and PDE4 inhibition with milrinone and rolipram 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 Mil + Rol (10 mM each), and after drug washout. B. Summary I–V relationships for the effects of Mil + Rol on right ventricular ICa,L. C. Summary ICa,L conductance density plots for the effects of Mil + Rol on right ventricular myocytes. Summary data are means 6 SEM; n = 8 ventricular myocytes; *P,0.05 vs. control by paired Student’s t-test. (TIF) References 1. Bender AT, Beavo JA (2006) Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev 58:488–520. 2. Omori K, Kotera J (2007) Overview of PDEs and their regulation. Circ Res 100:309–327. 3. Maurice DH, Palmer D, Tilley DG, Dunkerley HA, Netherton SJ, et al. (2003) Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system. Mol Pharmacol 64:533–546. 4. Fischmeister R, Castro LR, Abi-Gerges A, Rochais F, Jurevicius J, et al. (2006) Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases. Circ Res 99:816–828. 5. Movsesian MA (2002) PDE3 cyclic nucleotide phosphodiesterases and the compartmentation of cyclic nucleotide-mediated signalling in cardiac myocytes. Basic Res Cardiol 97 Suppl 1:I83–I90. 6. Zaccolo M (2011) Spatial control of cAMP signalling in health and disease. Curr Opin Pharmacol 11:649–655. 7. Patrucco E, Albergine MS, Santana LF, Beavo JA (2010) Phosphodiesterase 8A (PDE8A) regulates excitation-contraction coupling in ventricular myocytes. J Mol Cell Cardiol 49:330–333. 8. Fischmeister R, Hartzell HC (1991) Cyclic AMP phosphodiesterases and Ca2+ current regulation in cardiac cells. Life Sci 48:2365–2376. 9. Mongillo M, Tocchetti CG, Terrin A, Lissandron V, Cheung YF, et al. (2006) Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway. Circ Res 98:226–234. 10. Kerfant BG, Zhao D, Lorenzen-Schmidt I, Wilson LS, Cai S, et al. (2007) PI3K? is required for PDE4, not PDE3, activity in subcellular microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes. Circ Res 101:400–408. PDE Effects in Sinoatrial Node and Atrium Appendix S1 Supplemental materials and methods. (PDF) Table S1 Effects of IBMX on spontaneous action potential parameters in isolated mouse SAN myocytes. (PDF) Table S2 Effects of IBMX on stimulated action potential parameters in isolated mouse right atrial myocytes. (PDF) Table S3 Effects of EHNA on spontaneous action potential parameters in isolated mouse SAN myocytes. (PDF) Table S4 Effects of EHNA on stimulated action poten- tial parameters in isolated mouse right atrial myocytes. (PDF) Table S5 Effects of milrinone on spontaneous action potential parameters in isolated mouse SAN myocytes. (PDF) Table S6 Effects of milrinone on stimulated action potential parameters in isolated mouse right atrial myocytes. (PDF) Table S7 Effects of rolipram on spontaneous action potential parameters in isolated mouse SAN myocytes. (PDF) Table S8 Effects of rolipram on potential parameters in isolated myocytes. (PDF) Author Contributions stimulated action mouse right atrial Conceived and designed the experiments: RH AA RAR. Performed the experiments: RH AA CR GR RAR. Analyzed the data: RH AA CR GR RAR. Contributed reagents/materials/analysis tools: RAR. Wrote the paper: RH AA CR RR. 11. Verde I, Vandecasteele G, Lezoualc’h F, Fischmeister R (1999) Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytes. Br J Pharmacol 127:65–74. 12. Lakatta EG, Maltsev VA, Vinogradova TM (2010) A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart’s pacemaker. Circ Res 106:659–673. 13. DiFrancesco D (1993) Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol 55:455–472. 14. Irisawa H, Brown HF, Giles W (1993) Cardiac pacemaking in the sinoatrial node. Physiol Rev 73:197–227. 15. Mangoni ME, Nargeot J (2008) Genesis and regulation of the heart automaticity. Physiol Rev 88:919–982. 16. Mangoni ME, Couette B, Bourinet E, Platzer J, Reimer D, et al. (2003) Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity. Proc Natl Acad Sci U S A 100:5543–5548. 17. Zhang Z, Xu Y, Song H, Rodriguez J, Tuteja D, et al. (2002) Functional Roles of Cav1.3 (a1D) calcium channel in sinoatrial nodes: insight gained using gene- targeted mutant mice. Circ Res 90:981–987. 18. Nerbonne JM, Kass RS (2005) Molecular physiology of cardiac repolarization. Physiol Rev 85:1205–1253. 19. Zhang Z, He Y, Tuteja D, Xu D, Timofeyev V, et al. (2005) Functional roles of Cav1.3 (a1D) calcium channels in atria: insights gained from gene-targeted mutant mice. Circulation 112:1936–1944. 20. Springer J, Azer J, Hua R, Robbins C, Adamczyk A, et al. (2012) The natriuretic peptides BNP and CNP increase heart rate and electrical conduction by stimulating ionic currents in the sinoatrial node and atrial myocardium following activation of guanylyl cyclase-linked natriuretic peptide receptors. J Mol Cell Cardiol 52:1122–1134. PLOS ONE | www.plosone.org 11 October 2012 | Volume 7 | Issue 10 | e47652