Abstract-Cardiac beating arises from the spontaneous rhythmic excitation of sinoatrial (SA) node cells. Here we report that SA node pacemaker activity is critically dependent on Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII). In freshly dissociated rabbit single SA node cells, inhibition of CaMKII by a specific peptide inhibitor, autocamtide-2 inhibitory peptide (AIP, 10 mol/L), or by KN-93 (0.1 to 3.0 mol/L), but not its inactive analog, KN-92, depressed the rate and amplitude of spontaneous action potentials (APs) in a dose-dependent manner. Strikingly, 10 mol/L AIP and 3 mol/L KN-93 completely arrested SA node cells, which indicates that basal CaMKII activation is obligatory to the genesis of pacemaker AP. To understand the ionic mechanisms of the CaMKII effects, we measured L-type Ca 2ϩ current (I Ca, L ), which contributes both to AP upstroke and to pacemaker depolarization. (1 mol/L), but not its inactive analog, KN-92, decreased I Ca, L amplitude from 12Ϯ2 to 6Ϯ1 pA/pF without altering the shape of the current-voltage relationship. Both AIP and KN-93 shifted the midpoint of the steady-state inactivation curve leftward and markedly slowed the recovery of I Ca, L from inactivation. Similar results were observed using the fast Ca 2ϩ chelator BAPTA, whereas the slow Ca 2ϩ chelator EGTA had no significant effect, which suggests that CaMKII activity is preferentially regulated by local Ca 2ϩ transients. Indeed, confocal immunocytochemical imaging showed that active CaMKII is highly localized beneath the surface membrane in the vicinity of L-type channels and that AIP and KN-93 significantly reduced CaMKII activity. Thus, we conclude that CaMKII plays a vital role in regulating cardiac pacemaker activity mainly via modulating I Ca, L inactivation and reactivation, and local Ca 2ϩ is critically involved in these processes.

Recent studies have added complexities to the conceptual framework of cardiac beta-adrenergic receptor (beta-AR) signal transduction. Whereas the classical linear G(s)-adenylyl cyclase-cAMP-protein kinase A (PKA) signaling cascade has been corroborated for beta(1)-AR stimulation, the beta(2)-AR signaling pathway bifurcates at the very first postreceptor step, the G protein level. In addition to G(s), beta(2)-AR couples to pertussis toxin-sensitive G(i) proteins, G(i2) and G(i3). The coupling of beta(2)-AR to G(i) proteins mediates, to a large extent, the differential actions of the beta-AR subtypes on cardiac Ca(2+) handling, contractility, cAMP accumulation, and PKA-mediated protein phosphorylation. The extent of G(i) coupling in ventricular myocytes appears to be the basis of the substantial species-to-species diversity in beta(2)-AR-mediated cardiac responses. There is an apparent dissociation of beta(2)-AR-induced augmentations of the intracellular Ca(2+) (Ca(i)) transient and contractility from cAMP production and PKA-dependent cytoplasmic protein phosphorylation. This can be largely explained by G(i)-dependent functional compartmentalization of the beta(2)-AR-directed cAMP/PKA signaling to the sarcolemmal microdomain. This compartmentalization allows the common second messenger, cAMP, to perform selective functions during beta-AR subtype stimulation. Emerging evidence also points to distinctly different roles of these beta-AR subtypes in modulating noncontractile cellular processes. These recent findings not only reveal the diversity and specificity of beta-AR and G protein interactions but also provide new insights for understanding the differential regulation and functionality of beta-AR subtypes in healthy and diseased hearts.

In contrast to ␤ 1 -adrenoreceptor (␤ 1 -AR) signaling, ␤ 2 -AR stimulation in cardiomyocytes augments L-type Ca 2؉ current in a cAMP-dependent protein kinase (PKA)-dependent manner but fails to phosphorylate phospholamban, indicating that the ␤ 2 -AR-induced cAMP/PKA signaling is highly localized. Here we show that inhibition of G i proteins with pertussis toxin (PTX) permits a full phospholamban phosphorylation and a de novo relaxant effect following ␤ 2 -AR stimulation, converting the localized ␤ 2 -AR signaling to a global signaling mode similar to that of ␤ 1 -AR. Thus, ␤ 2 -AR-mediated G i activation constricts the cAMP signaling to the sarcolemma. PTX treatment did not significantly affect the ␤ 2 -ARstimulated PKA activation. Similar to G i inhibition, a protein phosphatase inhibitor, calyculin A (3 ؋ 10 ؊8 M), selectively enhanced the ␤ 2 -AR but not ␤ 1 -AR-mediated contractile response. Furthermore, PTX and calyculin A treatment had a non-additive potentiating effect on the ␤ 2 -AR-mediated positive inotropic response. These results suggest that the interaction of the ␤ 2 -AR-coupled G i and G s signaling affects the local balance of protein kinase and phosphatase activities. Thus, the additional coupling of ␤ 2 -AR to G i proteins is a key factor causing the compartmentalization of ␤ 2 -AR-induced cAMP signaling.

17phosphorylation by CaMKII and is essential to the relaxant effect of ␤-adrenergic stimulation. To determine the role of Thr 17 PLB phosphorylation, we investigated the dual-site phosphorylation of PLB in isolated adult rat cardiac myocytes in response to ␤ 1 -adrenergic stimulation or electrical field stimulation (0.1-3 Hz) or both. A ␤ 1 -adrenergic agonist, norepinephrine (10 ؊9 -10 (1-3). In the beating mammalian heart, ␤-adrenergic stimulation increases both PKA-and CaMKIImediated phosphorylation of Ser 16 and Thr 17 (4 -6). More recent studies have shown that the effects of ␤-adrenergic stimulation on PLB phosphorylation are mostly attributable to ␤ 1 -but not ␤ 2 -adrenergic receptor subtype (7-10).Over the last two decades, intensive studies have been focused on the physiological significance of the dual site phosphorylation of PLB. These previous studies in perfused hearts or in vivo have provided several lines of evidence leading to the concept that Ser 16 phosphorylation is a prerequisite for phosphorylation of Thr 17 and that Ser 16 phosphorylation is largely responsible for ␤-adrenergic modulation of cardiac relaxation (4, 5, 11, 12, 14 -18 phosphorylation is the dominant molecular event responsible for accelerated cardiac relaxation. However, in vitro studies in the isolated SR membranes have consistently indicated that Ser 16 and Thr 17 can be readily and independently phosphorylated by PKA and CaMKII, respectively, and that when both are phosphorylated, there is an additive interaction (20,21). The apparent discrepancy between in vivo and in vitro PLB phosphorylation is yet to be reconciled.To resolve this paradox and to further address the relative contribution of PKA-and CaMKII-mediated PLB phosphorylation in beat-to-beat cardiac functional modulation, individually we manipulated PKA activity, using ␤-adrenergic stimulation in quiescent rat ventricular myocytes, and CaMKII activity, by electrically pacing the myocytes at different stimulation frequency (0.1-3 Hz) in the absence of ␤-adrenergic stimulation. Both stimuli were also combined to explore possible interactions between PKA-and CaMKII-mediated signaling. Under those experimental conditions, we measured PLB phosphorylation at Ser 16 and Thr 17 as well as relaxation time of cell contraction. Here, we report our surprising findings that electrical stimulation alone increases CaMKII-dependent phosphorylation of PLB at Thr 17 in a frequency-dependent manner without altering PKA-mediated Ser 16 phosphorylation, that phosphorylation of Thr 17 is markedly enhanced by ␤-adrenergic stimulation in the electrically paced but not in quiescent myocytes, and that Thr 17 phosphorylation is associated with a significant relaxant effect, regardless of ␤-adrenergic stimulation.

A plausible determinant of the specificity of receptor signaling is the cellular compartment over which the signal is broadcast. In rat heart, stimulation of beta(1)-adrenergic receptor (beta(1)-AR), coupled to G(s)-protein, or beta(2)-AR, coupled to G(s)- and G(i)-proteins, both increase L-type Ca(2+) current, causing enhanced contractile strength. But only beta(1)-AR stimulation increases the phosphorylation of phospholamban, troponin-I, and C-protein, causing accelerated muscle relaxation and reduced myofilament sensitivity to Ca(2+). beta(2)-AR stimulation does not affect any of these intracellular proteins. We hypothesized that beta(2)-AR signaling might be localized to the cell membrane. Thus we examined the spatial range and characteristics of beta(1)-AR and beta(2)-AR signaling on their common effector, L-type Ca(2+) channels. Using the cell-attached patch-clamp technique, we show that stimulation of beta(1)-AR or beta(2)-AR in the patch membrane, by adding agonist into patch pipette, both activated the channels in the patch. But when the agonist was applied to the membrane outside the patch pipette, only beta(1)-AR stimulation activated the channels. Thus, beta(1)-AR signaling to the channels is diffusive through cytosol, whereas beta(2)-AR signaling is localized to the cell membrane. Furthermore, activation of G(i) is essential to the localization of beta(2)-AR signaling because in pertussis toxin-treated cells, beta(2)-AR signaling becomes diffusive. Our results suggest that the dual coupling of beta(2)-AR to both G(s)- and G(i)-proteins leads to a highly localized beta(2)-AR signaling pathway to modulate sarcolemmal L-type Ca(2+) channels in rat ventricular myocytes.

Background —Recent studies of β-adrenergic receptor (β-AR) subtype signaling in in vitro preparations have raised doubts as to whether the cAMP/protein kinase A (PKA) signaling is activated in the same manner in response to β 2 -AR versus β 1 -AR stimulation. Methods and Results —The present study compared, in the intact dog, the magnitude and characteristics of chronotropic, inotropic, and lusitropic effects of cAMP accumulation, PKA activation, and PKA-dependent phosphorylation of key effector proteins in response to β-AR subtype stimulation. In addition, many of these parameters and L-type Ca 2+ current ( I Ca ) were also measured in single canine ventricular myocytes. The results indicate that although the cAMP/PKA-dependent phosphorylation cascade activated by β 1 -AR stimulation could explain the resultant modulation of cardiac function, substantial β 2 -AR–mediated chronotropic, inotropic, and lusitropic responses occurred in the absence of PKA activation and phosphorylation of nonsarcolemmal proteins, including phospholamban, troponin I, C protein, and glycogen phosphorylase kinase. However, in single canine myocytes, we found that β 2 -AR–stimulated increases in both I Ca and contraction were abolished by PKA inhibition. Thus, the β 2 -AR–directed cAMP/PKA signaling modulates sarcolemmal L-type Ca 2+ channels but does not regulate PKA-dependent phosphorylation of cytoplasmic proteins. Conclusions —These results indicate that the dissociation of β 2 -AR signaling from cAMP regulatory systems is only apparent and that β 2 -AR–stimulated cAMP/PKA signaling is uncoupled from phosphorylation of nonsarcolemmal regulatory proteins involved in excitation-contraction coupling.

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Phospholamban is a critical regulator of sarcoplasmic reticulum Ca2+-ATPase and myocardial contractility. To determine the extent of cross signaling between Ca2+ and cAMP pathways, we have investigated the β-adrenergic-induced phosphorylation of Ser16 and Thr17 of phospholamban in perfused rat hearts using antibodies recognizing phospholamban phosphorylated at either position. Isoproterenol caused the dose-dependent phosphorylation of Ser16 and Thr17 with strikingly different half-maximal values (EC50 = 4.5 ± 1.6 and 28.2 ± 1.4 nmol/l, respectively). The phosphorylation of Ser16 induced by isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine correlated to increased cardiac relaxation ( r = 0.96), whereas phosphorylation of Thr17 did not. Elevation of extracellular Ca2+did not induce phosphorylation at Thr17; only in the presence of a submaximal dose of isoproterenol, phosphorylation at Thr17 increased eightfold without additional effects on relaxation rate. Thr17 phosphorylation was partially affected by ryanodine and was completely abolished in the presence of 1 μmol/l verapamil or nifedipine. The data indicate that 1) phosphorylation of phospholamban at Ser16 by cAMP-dependent protein kinase is the main regulator of β-adrenergic-induced cardiac relaxation definitely preceding Thr17 phosphorylation and 2) the β-adrenergic-mediated phosphorylation of Thr17 by Ca2+-calmodulin-dependent protein kinase required influx of Ca2+through the L-type Ca2+ channel.