Ca2+-clock-dependent pacemaking in the sinus node is impaired in mice with a cardiac specific reduction in SERCA2 abundance

Background: The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) pump is an important component of the Ca(2+)-clock pacemaker mechanism that provides robustness and flexibility to sinus node pacemaking. We have developed transgenic mice with reduced cardiac SERCA2 abundance (Serca2 KO) as a model for investigating SERCA2's role in sinus node pacemaking. Methods and Results: In Serca2 KO mice, ventricular SERCA2a protein content measured by Western blotting was 75% (P < 0.05) lower than that in control mice (Serca2 FF) tissue. Immunofluorescent labeling of SERCA2a in ventricular, atrial, sinus node periphery and center tissue sections revealed 46, 45, 55, and 34% (all P < 0.05 vs. Serca2 FF) lower labeling, respectively and a mosaic pattern of expression. With telemetric ECG surveillance, we observed no difference in basal heart rate, but the PR-interval was prolonged in Serca2 KO mice: 49 ± 1 vs. 40 ± 1 ms (P < 0.001) in Serca2 FF. During exercise, heart rate in Serca2 KO mice was elevated to 667 ± 22 bpm, considerably less than 780 ± 17 bpm (P < 0.01) in Serca2 FF. In isolated sinus node preparations, 2 mM Cs(+) caused bradycardia that was equally pronounced in Serca2 KO and Serca2 FF (32 ± 4% vs. 29 ± 5%), indicating no change in the pacemaker current, I(f). Disabling the Ca(2+)-clock with 2 μM ryanodine induced bradycardia that was less pronounced in Serca2 KO preparations (9 ± 1% vs. 20 ± 3% in Serca2 FF; P < 0.05), suggesting a disrupted Ca(2+)-clock. Mathematical modeling was used to dissect the effects of membrane- and Ca(2+)-clock components on Serca2 KO mouse heart rate and sinus node action potential. Computer modeling predicted a slowing of heart rate with SERCA2 downregulation and the heart rate slowing was pronounced at >70% reduction in SERCA2 activity. Conclusions: Serca2 KO mice show a disrupted Ca(2+)-clock-dependent pacemaker mechanism contributing to impaired sinus node and atrioventricular node function

A circadian clock in the sinus node mediates day-night rhythms in Hcn4 and heart rate

BACKGROUND Heart rate follows a diurnal variation, and slow heart rhythms occur primarily at night.OBJECTIVE The lower heart rate during sleep is assumed to be neural in origin, but here we tested whether a day-night difference in intrinsic pacemaking is involved.METHODS In vivo and in vitro electrocardiographic recordings, vagotomy, transgenics, quantitative polymerase chain reaction, Western blotting, immunohistochemistry, patch damp, reporter bioluminescence recordings, and chromatin immunoprecipitation were used.RESULTS The day-night difference in the average heart rate of mice was independent of fluctuations in average locomotor activity and persisted under pharmacological, surgical, and transgenic interruption of autonomic input to the heart. Spontaneous beating rate of isolated (ie, denervated) sinus node (SN) preparations exhibited a day-night rhythm concomitant with rhythmic messenger RNA expression of ion channels including hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4). In vitro studies demonstrated 24-hour rhythms in the human HCN4 promoter and the corresponding funny current. The day-night heart rate difference in mice was abolished by HCN block, both in vivo and in the isolated SN. Rhythmic expression of canonical circadian dock transcription factors, for example, Brain and muscle ARNT-Like 1 (BMAL1) and Cryptochrome (CRY) was identified in the SN and disruption of the local dock (by cardiomyocyte-specific knockout of Bmall) abolished the day-night difference in Hcn4 and intrinsic heart rate. Chromatin immunoprecipitation revealed specific BMAL1 binding sites on Hcn4, linking the local dock with intrinsic rate control.CONCLUSION The circadian variation in heart rate involves SN local dock-dependent Hcn4 rhythmicity. Data reveal a novel regulator of heart rate and mechanistic insight into bradycardia during sleep