Abstract-We recently showed that phosphoinositide-3-kinase-␥-deficient (PI3K␥ Ϫ/Ϫ ) mice have enhanced cardiac contractility attributable to cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca 2ϩ content and release but not L-type Ca 2ϩ current (I Ca,L ), demonstrating PI3K␥ locally regulates cAMP levels in cardiomyocytes. Because phosphodiesterases (PDEs) can contribute to cAMP compartmentation, we examined whether the PDE activity was altered by PI3K␥ ablation. Selective inhibition of PDE3 or PDE4 in wild-type (WT) cardiomyocytes elevated Ca 2ϩ transients, SR Ca 2ϩ content, and phospholamban phosphorylation (PLN-PO 4 ) by similar amounts to levels observed in untreated PI3K␥

The phosphorylation of the cardiac Ca 2؉ -release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) has been extensively characterized, but its functional consequence remains poorly defined and controversial. We have previously shown that RyR2 is phosphorylated by PKA at two major sites, serine 2030 and serine 2808, of which Ser-2030 is the major PKA site responding to ␤-adrenergic stimulation. Here we investigated the effect of the phosphorylation of RyR2 by PKA on the properties of single channels and on spontaneous Ca 2؉ release during sarcoplasmic reticulum Ca 2؉ overload, a process we have referred to as store overload-induced Ca 2؉ release (SOICR). We found that PKA activated single RyR2 channels in the presence, but not in the absence, of luminal Ca 2؉ . On the other hand, PKA had no marked effect on the sensitivity of the RyR2 channel to activation by cytosolic Ca 2؉ . Importantly, the S2030A mutation, but not mutations of Ser-2808, diminished the effect of PKA on RyR2. Furthermore, a phosphomimetic mutation, S2030D, potentiated the response of RyR2 to luminal Ca 2؉ and enhanced the propensity for SOICR in HEK293 cells. In intact rat ventricular myocytes, the activation of PKA by isoproterenol reduced the amplitude and increased the frequency of SOICR. Confocal line-scanning fluorescence microscopy further revealed that the activation of PKA by isoproterenol increased the rate of Ca 2؉ release and the propagation velocity of spontaneous Ca 2؉ waves, despite reduced wave amplitude and resting cytosolic Ca 2؉ . Collectively, our data indicate that PKA-dependent phosphorylation enhances the response of RyR2 to luminal Ca 2؉ and reduces the threshold for SOICR and that this effect of PKA is largely mediated by phosphorylation at Ser-2030. Ventricular tachycardia (VT)4 is the leading cause of sudden death, particularly in patients with heart failure (HF), but the molecular mechanisms underlying the high incidence of VT in HF are not completely understood (1). A major cause of VT is believed to be delayed afterdepolarizations, which are produced by spontaneous Ca 2ϩ release from the sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) during SR Ca 2ϩ overload (2-5), a process we referred to as store overload-induced Ca 2ϩ release (SOICR) (6, 7). Physical or emotional stresses, which activate the ␤-adrenergic receptor (AR)/protein kinase A (PKA) signaling pathway, are common triggers for SOICR. Marks' group has shown that RyR2 is phosphorylated by PKA at a single residue, Ser-2808 (10, 11), which was originally identified as a unique Ca 2ϩ -and calmodulin-dependent protein kinase II phosphorylation site (12,13 (23, 24). These results are seemingly inconsistent with those of in vitro studies. The reasons for this apparent discrepancy are unknown.Moderate modulation of RyR2 activity has been shown to have no sustained effect on stimulated SR Ca 2ϩ release due to the regulation of RyR2 by luminal Ca 2ϩ , a phenomenon often referred to as "SR auto-regulation" (25), but it does exert a ...