Distinct Patterns of Constitutive Phosphodiesterase Activity in Mouse Sinoatrial Node and Atrial Myocardium . . Rui Hua , Andrew Adamczyk , Courtney Robbins, Gibanananda Ray, Robert A. Rose* Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Abstract Phosphodiesterases (PDEs) are critical regulators of cyclic nucleotides in the heart. In ventricular myocytes, the L-type Ca2+ current (ICa,L) is a major target of regulation by PDEs, particularly members of the PDE2, PDE3 and PDE4 families. Conversely, much less is known about the roles of PDE2, PDE3 and PDE4 in the regulation of action potential (AP) properties and ICa,L in the sinoatrial node (SAN) and the atrial myocardium, especially in mice. Thus, the purpose of our study was to measure the effects of global PDE inhibition with Isobutyl-1-methylxanthine (IBMX) and selective inhibitors of PDE2, PDE3 and PDE4 on AP properties in isolated mouse SAN and right atrial myocytes. We also measured the effects of these inhibitors on ICa,L in SAN and atrial myocytes in comparison to ventricular myocytes. Our data demonstrate that IBMX markedly increases spontaneous AP frequency in SAN myocytes and AP duration in atrial myocytes. Spontaneous AP firing in SAN myocytes was also increased by the PDE2 inhibitor erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA), the PDE3 inhibitor milrinone (Mil) and the PDE4 inhibitor rolipram (Rol). In contrast, atrial AP duration was increased by EHNA and Rol, but not by Mil. IBMX also potently, and similarly, increased ICa,L in SAN, atrial and ventricular myocytes; however, important differences emerged in terms of which inhibitors could modulate ICa,L in each myocyte type. Consistent with our AP measurements, EHNA, Mil and Rol each increased ICa,L in SAN myocytes. Also, EHNA and Rol, but not Mil, increased atrial ICa,L. In complete contrast, no selective PDE inhibitors increased ICa,L in ventricular myocytes when given alone. Thus, our data show that the effects of selective PDE2, PDE3 and PDE4 inhibitors are distinct in the different regions of the myocardium indicating important differences in how each PDE family constitutively regulates ion channel function in the SAN, atrial and ventricular myocardium. Citation: Hua R, Adamczyk A, Robbins C, Ray G, Rose RA (2012) Distinct Patterns of Constitutive Phosphodiesterase Activity in Mouse Sinoatrial Node and Atrial Myocardium. PLoS ONE 7(10): e47652. doi:10.1371/journal.pone.0047652 Editor: Marcello Rota, Brigham & Women’s Hospital – Harvard Medical School, United States of America Received July 19, 2012; Accepted September 14, 2012; Published October 15, 2012 Copyright: 2012 Hua et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Canadian Institutes of Health Research (MOP 93718; http://www.cihr-irsc.gc.ca/e/193.html), the Heart and Stroke Foundation of Nova Scotia (http://www.hsf.ca/research/en/home), The Canada Foundation for Innovation (http://www.innovation.ca/en) and the Dalhousie Medical Research Foundation (http://www.dmrf.ca/en/home/default.aspx) to RAR who is a CIHR New Investigator. RH is supported by a postdoctoral fellowship from the Heart and Stroke Foundation of Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: robert.rose@dal.ca . These authors contributed equally to this work. Introduction Phosphodiesterases (PDEs) are phosphohydrolase enzymes that are responsible for the degradation of the cyclic nucleotides adenosine and guanosine 39,59 cyclic monophosphate (cAMP and cGMP) [1,2]. PDEs play critical roles in the modulation of cellular functions that depend on cAMP and cGMP in the heart, including electrical conduction, contractility, metabolism and transcription [3], by controlling the levels of these powerful signaling molecules in cells. PDEs exist in 11 families (PDE1-11) with several isoforms in each and are regulated by diverse mechanisms including phosphorylation, binding of cyclic nucleotides, calcium binding and protein-protein interactions [1]. With such a large number of families and isoforms PDE signaling is clearly complex and in this context it is now thought that PDEs are importantly involved in the compartmentation of cyclic nucleotide signaling whereby the subcellular localization of different PDE isoforms can lead to distinct spatial and temporal pools of cAMP and/or cGMP [3–6]. This can result in distinct roles for specific PDEs in different parts of the cell or in different conditions. Among the PDE families expressed in the heart [7] PDE2, PDE3 and PDE4 have been shown to contribute substantially to cyclic nucleotide regulation, especially in the context of modulat- ing ion channel function [3,4,8,9]. Numerous studies have demonstrated that the L-type Ca2+ current (ICa,L) is a critical target of regulation by PDEs in ventricular myocytes. Specifically, it is established that global PDE inhibition with the broad spectrum inhibitor 3-Isobutyl-1-methylxanthine (IBMX) potently increases basal ventricular ICa,L in mice and rats [8,10,11]. Interestingly, selective inhibition of PDE2, PDE3 or PDE4 alone has no effect on basal ICa,L; however combined inhibition of PDE3 and PDE4 does increase basal ICa,L and inhibition of PDE2, PDE3 and PDE4 increases basal ICa,L very similarly to IBMX [8,10,11]. Although the effects of PDE2, 3 and 4 inhibition on ICa,L have been well characterized in ventricular myocytes much less is known about the role of these PDE families in the sinoatrial node (SAN) and atria, particularly in mice, a very common model organism due to its use in studies incorporating genetic manipula- tions. PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e47652