Journal
CIRCULATION RESEARCH
Volume 87, Issue 9, Pages 760-767Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/01.RES.87.9.760
Keywords
sinoatrial node; L-type Ca2+ channel; Ca2+/calmodulin-dependent kinase II; local Ca2+ signaling
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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 Ca2+/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 mu mol/L), or by KN-93 (0.1 to 3.0 mu 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 mu mol/L AIP and 3 mu 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 Ca2+ current (I-Ca,I-L), which contributes both to AP upstroke and to pacemaker depolarization. KN-93 (1 mu mol/L), but not its inactive analog, KN-92, decreased I-Ca,I-L amplitude from 12+/-2 to 6+/-1 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,I-L from inactivation. Similar results were observed using the fast Ca2+ chelator BAPTA, whereas the slow Ca2+ chelator EGTA had no significant effect, which suggests that CaMKII activity is preferentially regulated by local Ca2+ 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 ATP 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,I-L inactivation and reactivation, and local Ca2+ is critically involved in these processes.
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