Introduction: Beat-to-beat variability in cycle length exists in spontaneously beating cardiac preparations of varying complexities from the level of the isolated whole heart to the single sinoatrial nodal cell (SANC). The nature of this variability is poorly characterised as are its fundamental physiological origins. Methods: Recordings of spontaneous electrical activity were made from hearts in vivo, during Langendorff-perfusion, and from single SANC. Heart rate variability (HRV) was calculated in the time- and frequency-domains at baseline and in response to pharmacological mediators that interfered with critical processes involved in automaticity (catecholamines, carbachol, ivabradine, zatebradine, ryanodine and thapsigargin). In addition, a novel 2D technique for imaging Ca2+ fluorescence in spontaneously beating, fluo4-AM loaded, patched single sinoatrial nodal cells was developed to investigate the biophysical behaviour of Ca2+ during pacemaking to see if variability in this was responsible for SANC HRV. Results: Under baseline, temperature-stable conditions, levels of HRV were greatest in vivo (human > rat). SANC exhibited slightly lower levels of HRV, whereas HRV levels expressed by Langendorff-perfused hearts were the least (rabbit > rat), although still comprised a significant proportion of the variability witnessed in vivo. Anaesthetising in vivo rabbits decreased HRV to levels similar to those seen in the Langendorff-perfused heart. HRV was decreased by catecholamines and by ryanodine/thapsigargin in the Langendorff heart. Conversely, HRV was increased by carbachol, ivabradine, zatebradine and ryanodine in SANC. Heart rate changes had a marked effect on levels of HRV. 2D Ca2+ imaging of SANC showed that diastolic local Ca2+ releases (LCRs) occurred earlier than previously thought, with early LCRs having characteristics that were distinct from later LCRs. Mean time of occurrence of all the LCRs within a given diastole closely predicted the duration of the cycle. The rate of restitution of the whole cell Ca2+ transient (used as a surrogate for the pumping function of SERCA) in turn closely predicted the mean time of occurrence of LCRs. Tight synchronisation of the electrical activity of the cell with the biophysical behaviour of Ca2+ appeared to predict shorter cycle lengths. Isoprenaline increased LCR amplitude, though did not increase LCR number, size or duration. Isoprenaline caused LCRs to occur earlier, and synchronised their occurrence and the rate of pumping of Ca2+ back into the sarcoplasmic reticulum. Finally, LCRs were found to preferentially recur in certain regions of the cell, dubbed hotspots. Isoprenaline favoured hotspot production. Conclusion: Whilst greatest in vivo, significant HRV exists in spontaneously beating cardiac preparations devoid of a functioning autonomic nervous system. Studies in SANC indicate that the origin of this is likely to be variability in release of LCRs from the SR via ryanodine receptors. This in turn is controlled by SR refilling kinetics via SR Ca2+ pumping. The coupled system of membrane- and Ca2+-pacemaker clocks are so heavily intertwined that myriad factors will come to bear on generating such variability, including the amount of Ca2+ available for pumping and the phosphorylation state of key proteins, to the extent that variability in no one process can take the credit for generating such HRV.

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