Sinoatrial Node Pacemaker Activity Requires Ca ²⁺ /Calmodulin-Dependent Protein Kinase II Activation

Abstract

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.

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

“…Surprisingly, I Ca facilitation was completely abolished in SR-AIP. In addition, I Ca recovery from inactivation, a process slowed by AIP [36,37] and by the synthetic CaMKII inhibitor KN-93 [35], was also markedly slowed in SR-AIP. Therefore our results suggest that the CaMKII inhibiting peptide located in the SR membrane inhibits CaMKIIdependent phosphorylation of the L-type Ca 2+ channel.…”

“…Both processes depend on CaMKII [21,22,24,36,37] but if the AIP moieties in the fusion protein anchored in the SR membrane cannot reach the sarcolemma these processes should be unaltered in SR-AIP mice. To rule out secondary effects on I Ca facilitation due to changes in intracellular Ca 2+ handling, we performed these experiments in the presence of 10 mM EGTA, therefore abolishing intracellular Ca 2+ transients.…”

Normal Heart Rhythm is Initiated and Regulated by an Intracellular Calcium Clock Within Pacemaker Cells

“…Interestingly, in rabbit SANC LCRs predominantly occur within a time window of ~20% of the cycle length before an excitation [10,14], i.e., exactly where "1:1 entrainment zone" was originally discovered [23]. Also, arrhythmic beating showing various alternation patterns can be observed in rabbit SANC when the SR clock is slowed by chelation of intracellular Ca 2+ [25], by disabling Ca 2+ release by ryanodine [7] or by inhibition of PKA signaling [5,26].…”

Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model

“…TN-XXL expression, however, caused a reduced maximal heart rate under maximal adreneric stimulation, thus reducing the overall dynamic range of heart rate regulation. It is possible that overexpression of TN-XXL buffers Ca 2 + in SAN pacemaker cells, which is required to increase the slope of the pacemaker potential 41 and is utilized to support the physiological increase in heart rate. In line with this hypothesis it has been shown that chelating SAN Ca 2 + by EGTA 41 , BAPTA 41 or the Ca 2 + indicator Indo-1 (refs 42,43) significantly reduces the slope of diastolic depolarization and beating rate of sinoatrial pacemaker cells.…”