We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before beta-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after beta-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon beta-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon beta-adrenergic stimulation.

K201 (JTV519), a benzothiazepine derivative, has been shown to possess anti-arrhythmic and cardioprotective properties, but the mechanism of its action is both complex and controversial. It is believed to stabilize the closed state of the RyR2 (cardiac ryanodine receptor) by increasing its affinity for the FKBP12.6 (12.6 kDa FK506 binding protein) [Wehrens, Lehnart, Reiken, Deng, Vest, Cervantes, Coromilas, Landry and Marks (2004) Science 304, 292-296]. In the present study, we investigated the effect of K201 on spontaneous Ca2+ release induced by Ca2+ overload in rat ventricular myocytes and in HEK-293 cells (human embryonic kidney cells) expressing RyR2 and the role of FKBP12.6 in the action of K201. We found that K201 abolished spontaneous Ca2+ release in cardiac myocytes in a concentration-dependent manner. Treating ventricular myocytes with FK506 to dissociate FKBP12.6 from RyR2 did not affect the suppression of spontaneous Ca2+ release by K201. Similarly, K201 was able to suppress spontaneous Ca2+ release in FK506-treated HEK-293 cells co-expressing RyR2 and FKBP12.6. Furthermore, K201 suppressed spontaneous Ca2+ release in HEK-293 cells expressing RyR2 alone and in cells co-expressing RyR2 and FKBP12.6 with the same potency. In addition, K201 inhibited [3H]ryanodine binding to RyR2-wt (wild-type) and an RyR2 mutant linked to ventricular tachycardia and sudden death, N4104K, in the absence of FKBP12.6. These observations demonstrate that FKBP12.6 is not involved in the inhibitory action of K201 on spontaneous Ca2+ release. Our results also suggest that suppression of spontaneous Ca2+ release and the activity of RyR2 contributes, at least in part, to the anti-arrhythmic properties of K201.

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 ...

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Micro-needle electrodes (MEs) have attracted more and more attention for monitoring physiological electrical signals, including electrode-skin interface impedance (EII), electromyography (EMG) and electrocardiography (ECG) recording. A magnetization-induced self-assembling method (MSM) was developed to fabricate a microneedle array (MA). A MA coated with Ti/Au film was assembled as a ME. The fracture and insertion properties of ME were tested by experiments. The bio-signal recording performance of the ME was measured and compared with a typical commercial wet electrode (Ag/AgCl electrode). The results show that the MA self-assembled from the magnetic droplet array under the sum of gravitational surface tension and magnetic potential energies. The ME had good toughness and could easily pierce rabbit skin without being broken or buckling. When the compression force applied on the ME was larger than 2 N, ME could stably record EII, which was a lower value than that measured by Ag/AgCl electrodes. EMG signals collected by ME varied along with the contraction of biceps brachii muscle. ME could record static ECG signals with a larger amplitude and dynamic ECG signals with more distinguishable features in comparison with a Ag/AgCl electrode, therefore, ME is an alternative electrode for bio-signal monitoring in some specific situations.