Abstract-It has long been recognized that activation of sympathetic ␤-adrenoceptors (␤-ARs) increases the spontaneous beating rate of sinoatrial nodal cells (SANCs); however, the specific links between stimulation of ␤-ARs and the resultant increase in firing rate remain an enigma. In the present study, we show that the positive chronotropic effect of ␤-AR stimulation is critically dependent on localized subsarcolemmal ryanodine receptor (RyR) Ca 2ϩ releases during diastolic depolarization (CRDD). Specifically, isoproterenol (ISO; 0.1 mol/L) induces a 3-fold increase in the number of CRDDs per cycle; a shift to higher CRDD amplitudes (from 2.00Ϯ0.04 to 2.17Ϯ0.03 F/F 0 ; PϽ0.05 [F and F 0 refer to peak and minimal fluorescence]); and an increase in spatial width (from 3.80Ϯ0.44 to 5.45Ϯ0.47 m; PϽ0.05). The net effect results in an augmentation of the amplitude of the local preaction potential subsarcolemmal Ca 2ϩ transient that, in turn, accelerates the diastolic depolarization rate, leading to an increase in SANC firing rate. When RyRs are disabled by ryanodine, ␤-AR stimulation fails to amplify subsarcolemmal Ca 2ϩ releases, fails to augment the diastolic depolarization rate, and fails to increase the SANC firing rate, despite preserved ␤-AR stimulation-induced augmentation of L-type Ca 2ϩ current amplitude. Thus, the RyR Ca 2ϩ release acts as a switchboard to link ␤-AR stimulation to an increase in SANC firing rate: recruitment of additional localized CRDDs and partial synchronization of their occurrence by ␤-AR stimulation lead to an increase in the heart rate.

Abstract-The rate of spontaneous diastolic depolarization (DD) of sinoatrial nodal cells (SANCs) that triggers recurrent action potentials (APs) is a fundamental aspect of the heart's pacemaker. Here, in experiments on isolated SANCs, using confocal microscopy combined with a patch clamp technique, we show that ryanodine receptor Ca 2ϩ release during the DD produces a localized subsarcolemmal Ca 2ϩ increase that spreads in a wavelike manner by Ca 2ϩ -induced Ca 2ϩ release and produces an inward current via the Na ϩ -Ca 2ϩ exchanger (NCX , an interaction that generates inward current, is involved in spontaneous SANC beating and thus in SANC pacemaker function. However, the characteristics of this localized pre-AP release in isolated SANCs have not been determined. In addition, the extent to which the forward mode of electrogenic NCX activated by Ca 2ϩ release from SR contributes to the DD and modulates the SANC beating rate is unknown.Therefore, the present study specifically characterized localized pre-AP Ca 2ϩ release in isolated rabbit SANCs, characterized the current generated by its activation of the NCX, and determined the effect of this coordinated operation of ryanodine receptors (RYRs) and NCX on spontaneous SANC beating rate. Our findings indicate that in SANC pre-AP Ca 2ϩ releases are locally propagating Ca 2ϩ waves resulting from ryanodine-sensitive CICR. A negative chronotropic effect of ryanodine is accompanied by disappearance of the localized pre-AP Ca 2ϩ releases. The present results also Original

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.

No abstract