PDE Effects in Sinoatrial Node and Atrium Figure 1. Quantitative mRNA expression of PDE 2, 3 and 4 subtypes in mouse SAN, right atrium and right ventricle. A. Expression of PDE2A, PDE3A, PDE3B, PDE4A, PDE4B and PDE4D are shown relative to GAPDH for the SAN, right atrium (RA) and right ventricular free wall (RV). B. SAN samples were distinguished from RA samples by the characteristic pattern of expression of HCN4 and ANP in these regions. SAN tissue shows high expression of HCN4 and low expression of ANP whereas the RA shows very low HCN4 expression and very high ANP expression. Note scale break on Y-axis. Data are means 6 SEM; n = 5 SAN trials, 5 right atrial appendage trials and 3 right ventricular free wall trials; *P,0.05; {P,0.001 by two way ANOVA with Tukey’s posthoc test. doi:10.1371/journal.pone.0047652.g001 The SAN contains the specialized pacemaker myocytes whose spontaneous activity is responsible for determining heart rate [12]. Spontaneous action potentials (APs) in these SAN myocytes are characterized by the presence of a diastolic depolarization (DD), during which the SAN myocyte gradually depolarizes until the threshold for an AP is reached [12–14]. Several ionic currents and mechanisms contribute to the generation of the DD including ICa,L [15]. Consistent with this hypothesis, it has recently been demonstrated that two forms of L-type Ca2+ channel, CaV1.2 and CaV1.3, contribute to total ICa,L in SAN myocytes, in contrast to ventricular myocytes which only express CaV1.2 [16,17]. CaV1.3 channels activate at more negative membrane potentials than CaV1.2 enabling them to contribute prominently to the generation of the DD and thereby to the frequency of spontaneous AP firing and heart rate control. ICa,L is also a critical determinant of AP morphology in working atrial myocytes [18]. Like SAN myocytes, CaV1.2 and CaV1.3 both contribute to total ICa,L in atrial myocytes [19]. Given the unique physiology of the SAN and atria it is possible that PDE regulation of AP properties and ion channels, including ICa,L, is different in these tissues in comparison to the ventricles. The purpose of this study was to determine the roles of PDE2, PDE3 and PDE4 in regulating AP properties in mouse SAN and atrial myocytes. We also measured the effects of broad spectrum and family specific PDE inhibitors on basal ICa,L in SAN and atrial myocytes in comparison to ventricular myocytes. This work represents the first direct comparison of these inhibitors on ICa,L in these three regions of the myocardium in mice. Our data demonstrate that the patterns of constitutive PDE activity are distinct between SAN, atrial and ventricular myocytes, further adding to the complexity of PDE signaling in the heart. Materials and Methods Ethics Statement This study utilized male wildtype C57Bl/6 mice (Charles River) between the ages of 10–14 weeks. Experimental procedures were in accordance with the regulations of The Canadian Council on Animal Care and were approved by The Dalhousie University Committee on Laboratory Animals. In all experiments, mice were anesthetized by isoflurane inhalation and cervically dislocated before hearts were removed. An expanded Materials and Methods section is available in Appendix S1. Experimental Approaches Quantitative mRNA expression for PDE2A, PDE3A, PDE3B, PDE4A, PDE4B and PDE4D in mouse SAN, right atrium and right ventricular free wall was performed using methods we have described previously (see also Appendix S1) [20]. These specific PDE isoforms were selected because our electrophysiological experiments are focused on the PDE2, 3 and 4 families and prior studies [7,11] have shown that the isoforms we measured are expressed in whole ventricular tissue and ventricular myocytes. Mouse SAN, right atrial and right ventricular myocytes were isolated for patch-clamp recordings using procedures we have described previously [20–23]. Spontaneous and stimulated action potentials (APs) were recorded using the perforated patch-clamp technique in current clamp mode. ICa,L was recorded in the whole cell patch-clamp configuration. All recordings were performed at room temperature (22–23uC). The solutions and electrophysio- logical protocols for these experiments are provided in Appen- dix S1. Statistics All summary data are presented as means 6 SEM. Data were analyzed by one or two way ANOVA with Tukey’s posthoc analysis or paired Student’s t-test. P,0.05 was considered significant. Results Region specific mRNA expression of PDE2, 3 and 4 subtypes in the heart Amongst the PDE2, 3 and 4 families, prior studies have shown that the PDE2A, PDE3A, PDE3B, PDE4A, PDE4B and PDE4D subtypes are expressed in whole ventricular myocardium and/or isolated ventricular myocytes in rodents [7,11]. The relative expression of these PDE isoforms in the SAN and right atrial PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47652