Pacemaking in the sino-atrial (SA) node is thought to be determined by sequential activation and deactivation of a variety of ionic currents. Evidence has accumulated in recent years supporting the possibility that this pacemaker activity might be influenced by changes in cytosolic calcium. Calcium entry and extrusion across the surface membrane, as well as calcium release and uptake by the sarcoplasmic reticulum (SR), are expected to underlie the changes in cytosolic calcium. Detailed mechanisms by which pacemaker activity might be influenced by cytosolic calcium remain to be determined, though one possibility that has received recent support is that localised changes in calcium resembling calcium sparks may be particularly important. For example, calcium spark-like events triggered by calcium entry through T-type calcium channels and giving rise to depolarising sodium-calcium exchange current might speed the early development of the upstroke of the action potential in cat SA node cells (Hüser et al. 2001). In addition, the positive chronotropic effects of β-adrenoceptor agonists have been associated with a ryanodine-sensitive increase in the frequency of spark-like events during diastolic depolarisation, and to changes in the characteristics of these events in rabbit SA node cells (Vinogradova et al. 2002). Evidence for the presence of ryanodine receptors, and for their involvement in β-adrenoceptor-mediated increases in rate of beating in guinea-pig SA node cells, has also been reported (Rigg et al. 2000).
It remains possible that ionic currents other than electrogenic sodium-calcium exchange current may also be influenced by changes in cytosolic calcium. In addition, while spark-like events may well play important roles in pacemaking, more sustained and/or widespread changes in cytosolic calcium could modulate a variety of currents that contribute to pacemaker activity. The aim of this presentation is to discuss evidence gathered from guinea-pig SA node cells concerning the importance of cytosolic calcium during pacemaker activity. In some experiments, photometric techniques with indo-1 as calcium indicator were used to measure calcium changes, while in others confocal microscopy (either linescan or Nipkow disk methods) and fluo-4 fluorescence were employed. Permeabilised patch-clamp techniques (with amphotericin in the pipette solution) were consistently applied for electrical recording. Spark-like activity was detected as separable events in approximately 30 % of beating SA node cells. β-Adrenoceptor stimulation increased the frequency and amplitude of the spark-like events. Positive chronotropic effects of β-adrenoceptor stimulation were also associated with increases in the amplitude and rate of decay of whole-cell calcium transients. The If current activated by hyperpolarization was suppressed by loading cells with the calcium chelator BAPTA and by the calmodulin antagonist W7, though not by the calmodulin-dependent kinase inhibitor KN93. This inhibitor did, however, appear to reduce L-type calcium currents and the delayed rectifier potassium currents IKr and IKs. These delayed rectifier potassium currents were also reduced by chelating cytosolic calcium with BAPTA, by reducing SR calcium release with ryanodine and by reducing calcium entry using nifedipine. Reducing extracellular sodium led to reduced rate of beating in the first minute of exposure but to increases in the amplitude of calcium transients measured either with indo-1 photometric techniques or with fluo-4 and confocal microscopy.
These observations are interpreted as supporting the importance of cytosolic calcium in influencing pacemaker activity. It appears that the actions of calcium are complex and may involve modulation, perhaps by several different mechanisms, of many of the ionic currents contributing to pacemaker activity. While spark-like events are thought to play an important role, more sustained and/or widespread effects of cytosolic calcium may also influence pacemaker activity.
This work was supported by The Wellcome Trust and the British Heart Foundation.