Putting the pacemaker channel through its paces to build a better biological pacemaker

Pacemaker channels, as has been appreciated since they were first described, have a most unusual and complex set of properties (DiFrancesco & Borer, 2007). This complexity belies their role in the sinoatrial (SA) node, which is deceivingly simple: to enable nodal myocytes to beat in an efficient and cyclic AMP-sensitive manner. This singularity of purpose has made them a preferred choice to provide a pacing boost to cells with little inclination to beat on their own, in the form of a biological pacemaker (Rosen et al. 2007, 2008), as well as a target for drugs that inhibit heart rate specifically (DiFrancesco & Borer, 2007). Pacemaker channels are also known as ‘funny’ or ‘hyperpolarization-activated cyclic nucleotide-modulated’ (HCN) channels in reference to their uncommon characteristics. Of the four mammalian HCN isoforms (HCN1–4), the HCN2 channel has been favoured to deliver pacing behaviour because the rate at which it opens and closes is faster than the HCN3 and HCN4 isoforms, which should produce appropriate beating rates easily. HCN2 channel opening, unlike HCN1, is strongly facilitated by cyclic AMP and, thus, can endow myocytes with sensitivity to autonomic and hormonal input. Nevertheless, even HCN2 channels may not provide rates that are high enough when delivered in vivo. In dogs whose SA or atrioventricular node conduction was suppressed, HCN2 gene delivery to the left ventricular conduction system significantly raised heart rates to ∼50–60 beats min−1 and approached 90 beats min−1 upon stimulation by adrenaline (Plotnikov et al. 2004; Bucchi et al. 2006). Nonetheless, these rates are still low compared to rates before conduction was compromised. Similar results were found in mice following induction of complete atrioventricular block and HCN2 gene delivery to the left ventricle (Piron et al. 2008). Utilization of the HCN1 isoform, which opens and closes significantly faster than HCN2, might confer a faster rate but its function is not strongly modulated by cAMP. Enter the chimeric channel HCN212, comprising the HCN1 pore-forming region and N- and C-terminal portions of HCN2. In this chimera, the desired functions are coupled: the fast opening and closing action of the HCN1 isoform and the sensitivity to cAMP associated with HCN2 (Wang et al. 2001). However, when this chimera was expressed in the left bundle branch in the dog model, the resultant heart rate was too fast (> 220 beats min−1) and it was highly irregular, with variable stretches of tachycardia and prolonged pauses interspersed (Plotnikov et al. 2008). Why did HCN212 produce this irregular beating pattern? In this issue of The Journal of Physiology, Zhao et al. (2009) exploit the advantages of isolated cardiomyocytes to explore this question. HCN2 and HCN212 channels were expressed in myocytes isolated from the rat ventricle from which the action potential, pacemaker current and other electrical elements could be examined directly and easily. Importantly, they found that the beating induced by each of the constructs was similar to that produced in vivo, demonstrating the utility of this surrogate system to investigate comparative features of the two HCN channels and the molecular mechanisms underlying the different patterns of beating induced. In particular, no differences in the voltage dependence of activation or HCN current density between HCN2 and HCN212 was observed, but the rates of opening and closing were faster in HCN212-expressing myocytes – as expected. Intriguingly, both channel forms, when stimulated at rates mimicking regular beating, exhibited significantly slowed rates of opening and closing. This was accompanied by an increase in the amplitude of ‘instantaneous current’, which flows through channels that do not close before the subsequent stimulation. The amount of instantaneous current is greater for HCN2 because it closes more slowly than HCN212. It was suggested that a greater proportion of open pacemaker channels would be available following repolarization in cells expressing HCN2, which would then contribute to a larger instantaneous current that stabilizes beating rate. Curiously, in mice, sinus pauses are also a consequence of knocking out the HCN4 gene (Herrmann et al. 2007) and occur following exercise or isoprenaline administration when an HCN4 mutant channel that lacks sensitivity to cAMP but is more easily opened by hyperpolarization is overexpressed (Harzheim et al. 2008). Together, the findings suggest that pacemaker channels confer sufficiently fast and stable beating rates, but only if they are present in the appropriate amounts and possess the right balance of kinetic properties. In models of SA node firing that incorporate precise descriptions of the very slow and unwieldy kinetic features of pacemaker channels, HCN2 and HCN4 channels produce regular beating whereas HCN1 channels have a greater propensity to produce irregular beating (Mannikko et al. 2005; Elinder et al. 2006) or even, paradoxically, to slow the beating rate (Azene et al. 2005). Further modelling and experimentation is needed to explore the complex kinetics and instantaneous current associated with each pacemaker channel, and to determine how these might affect other ionic elements in cardiac myocytes, particularly in the context of altered rhythmicity. These approaches can be combined and applied in a rigorous yet facile manner using the isolated cell system developed by Zhao et al. Through development of this in vitro cardiomyocyte system to effectively screen potential pacemaker channel candidates for eventual testing in vivo and to understand their impact on pacing at the molecular level, Zhao et al. have greatly aided and accelerated the building of a better biological pacemaker appropriately responsive to the changing needs of the body.

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