PDE Effects in Sinoatrial Node and Atrium Figure 8. Effects of the PDE3 inhibitor milrinone 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 Mil (10 mM) and following Mil washout. B. Summary I–V relationships for the effects of Mil on SAN myocyte ICa,L. C. Summary ICa,L conductance density plots for the effects of Mil in SAN myocytes. D. Representative ICa,L recordings in right atrial myocytes in control conditions, in the presence of Mil (10 mM), and following Mil washout. E. Summary I–V relationships for the effects of Mil on right atrial myocyte ICa,L. F. Summary ICa,L conductance density plots for the effects of Mil 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.g008 and/or enzymatic activity. Our mRNA measurements were done at the tissue level thus, in addition to myocytes, other cell types, such as cardiac fibroblasts, would be expected to be present in these samples and may contribute to total PDE mRNA levels. Further studies will therefore be required to better understand the basis for the differences in effects of PDE inhibitors in each region of the myocardium. As our study represents the first direct comparison of different PDE inhibitors on electrophysiological responses in SAN, atrial and ventricular myocytes in mice it is useful to compare our data with those reported in studies that have mainly used other species. Few studies have characterized the effects of PDE inhibitors in the specialized SAN; however, one recent report [34] did demonstrate the effects of various PDE inhibitors on spontaneous AP frequency in rabbit SAN myocytes. This study showed that PDE3 was the major constitutively active PDE in the basal state in rabbit, with Mil increasing AP frequency almost as effectively as IBMX. Other PDE inhibitors, including EHNA and Rol had much smaller effects. This is different from what we have observed in the mouse in which Mil and Rol each had similar stimulatory effects on AP frequency and where the stimulatory effect of EHNA, although smaller than Mil and Rol, was still robust. Other studies have also shown that PDE3 inhibitors increase SAN AP frequency in rabbits [35] and guinea pigs [36]; however, these studies did not compare these effects with inhibitors of other PDE families. Our data showing that PDE3 and PDE4 inhibitors can similarly increase AP frequency and ICa,L in mouse SAN myocytes are consistent with studies showing these inhibitors also increase beating rate in mouse and rat atrial preparations [37]. Several studies have measured the effects of selective PDE inhibitors on ICa,L in human atrial myocytes and these findings can be compared to our observations in mouse right atrial myocytes. Similar to our findings in mouse atrial myocytes, inhibition of PDE2 with EHNA potentiates basal ICa,L in human atrial myocytes [27] indicating that PDE2 regulates basal ICa,L in both mouse and human atrium. In contrast, whereas we found no effect of Mil on basal ICa,L in mouse atrial myocytes, PDE3 inhibition does increase basal ICa,L in human atrial myocytes [28,38]. Recently, PDE4 has also been shown to regulate basal cAMP levels in human atrial myocytes using a fluorescence resonance energy transfer based reporter system [39] suggesting that PDE4 likely regulates basal ICa,L in human atrium as it does in mice. This would be consistent with data indicating that the role of PDE4 in cAMP regulation is conserved between human and rodent hearts [31]. Our experiments have focused on the PDE2, 3 and 4 families and on the role of ICa,L in mediating changes in AP properties in SAN and atrial myocytes. This was done in order to facilitate comparisons and highlight differences with the effects of PDE inhibitors on ventricular ICa,L. Although the changes in AP properties we observed in SAN and atrial myocytes correlated very well with changes in ICa,L it is likely that other ionic mechanisms contribute as well. For example, the hyperpolarization-activated current (If) contributes prominently to the DD in SAN myocytes and is well known to be directly modulated by cAMP [13,15]. We have shown that PDE3 inhibition with Mil increases basal If in mouse SAN myocytes [20] and it has also been shown that IBMX increases If in rabbit SAN myocytes [40]. Also, the slope of the DD in SAN myocytes is partly determined by Ca2+ release from the SR and a subsequent activation of the Na+-Ca2+ exchanger (INCX) [12]. This PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e47652