Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C015

Oral Communications

Circadian rhythm in heart rate is due to an intrinsic circadian clock in the sinus node

Y. Wang1, A. Johnsen2, S. Logantha1, S. Olieslagers4, H. Ni1, C. Cox1, A. Bucchi3, S. Wegner1, B. Bano-Otalora1, C. Petit1, E. Gill1, N. Ashton1, G. Hart1, H. Zhang1, E. J. Cartwright1, U. Wisloff2, P. Martins4, D. DiFrancesco3, H. Dobrzynski1, H. Piggins1, M. R. Boyett1, A. D'Souza1

1. The University of Manchester, Manchester, United Kingdom. 2. Norwegian University of Science and Technology, Trondheim, Norway. 3. University of Milan, Milan, Italy. 4. Maastricht University, Maastricht, Netherlands.

There is a circadian rhythm in the resting heart rate (HR), which is slower at night. In addition, bradyarrhythmias primarily occur at night. We hypothesised that an intrinsic mechanism in the pacemaker of the heart, the sinus node (SAN), is involved. In conscious C57BL/6J mice maintained during a 12 h light-12 h dark cycle, there was a circadian rhythm in HR measured by telemetry: the HR was 61 beats/min slower at the start of sleep (light) period (Zeitgeber time, ZT 0), as compared to the start of awake (dark) period, ZT12 (P<0.05). There was no correlation between HR and activity by telemetry measurement. As a further test, a 1 h light pulse was given at the start of the awake period; activity ceased but the HR remained relatively high. We conclude that the circadian rhythm in HR is not activity dependent. Furthermore, there was a circadian rhythm in the intrinsic HR measured either using the isolated Langendorff-perfused heart or the isolated SAN: the HR was 97 and 71 beats/min slower at ZT 0 as compared to ZT12 (P<0.05) respectively. This suggests that an intrinsic mechanism in the heart is at least contributing to the circadian rhythm in HR. This is likely to be either the Ca2+ clock or the membrane clock responsible for pacemaking. To study the Ca2+ clock, the number of Ca2+ sparks and the diastolic Ca2+ level were significantly reduced at ZT 12 compared with ZT 0. Furthermore, in isolated SAN preparations, the change in HR caused by 2 mM ryanodine (incapacitates the Ca2+ clock) was not significant between ZT 0 and ZT 12. To study the membrane clock, patch clamp experiments were conducted on SAN cells isolated at the two time points. The key pacemaker current, If (funny current), was measured during 5 s hyperpolarizing pulses from a holding potential of -35 mV. If (at -125 mV) at ZT 2 was 17.8±2.8 pA/pF, whereas at ZT 14 it was 35.0±6.3 pA/pF (P<0.05). This change could explain or at least contribute to the circadian rhythm in HR. Consistent with this, on application of 2 mM Cs+ to block If, the circadian rhythm in HR in the isolated SAN was abolished. Furthermore, in the conscious mouse, injection of 6mg/kg ivabradine to block If abolished the circadian rhythm in HR. HCN channels are responsible for If. SAN biopsies were collected at ZT 0 and ZT 12 and the expression level of HCN4 (principal ion channel responsible for If) was measured at the mRNA level by qPCR and the protein level by Western blot. HCN4 mRNA was 89% higher at ZT 0 compared to ZT 12, whereas HCN4 protein was 49% lower (P<0.05); although the changes are discordant, a time lag of hours is expected between mRNA and protein, and the protein change is consistent with the change in If density. Further experiments showed that HCN4 is directly regulated by a local circadian clock in the SAN. It is concluded that a circadian rhythm in HCN4 and If driven by an intrinsic circadian clock in the SAN at least contributes to the circadian rhythm in HR.

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