The attrioventricular (AV) node co-ordinates the normal sequence of beating of the heart's upper and lower chambers (the atria and ventricles). We have a long standing interest in the study of AV myocytes and the role they play in arrhythmia (the disruption in the timing of cardiac muscle contraction) and excitation-contraction coupling (e.g. Doggrell & Hancox 2003, 2005; Crampin et al 2006; James & Hancox 2007). We are continuing our interest in this structure as well as in the mechanisms of action of anti-arrhythmic drugs such as amiodarone (Zhang et al 2010) and ranolazine (Hancox & Doggrell 2010).
We are interested in the role of ion channels and acidosis in the regulation of electrical activity in AV node myocytes. One of the principle ion channels we are interested in is the hERG channel, a voltage gated K+ channel that is responsible for the IKR current, one of the many currents that make up the cardiac action potential (cAP). The IKR current is important in regulating ventricular action potential duration and so the QT interval of the EEG. Its disruption is strongly associated with both congential and drug-induced arrythmias such as long- and short-QT syndrome (e.g. Modell & Lehmann 2006, O'Leary & Hancox 2010).
Acidosis is a major pathological modifier of cardiac ion channel function, including the hERG channel. It has been shown that hERG channels protect against unwanted premature stimulation at the end of the cAP by allowing a outward leak of K+ ions in the late phase of membrane repolarisation and part of our recent work has been to show that acidosis reduces such protection (Du et al 2010). We have also shown that the inhibition of the hERG channel is modulated by extracellular rather than intracellular acidosi (Du et al 2011a) and that there is a differential effect of acidosis on hERG subtypess, with channels containing the hERGb subunit being more susceptible to acidosis than those without it (Du et al 2011b). Little is known about the relative distributions of these isoforms of the hERG channel, but it could be an important factor in determining the severity of arrhythmia during pathological acidosis.
The coupling of the cardiac action potential to the contraction of the cardiac muscle is of crucial importance to the proper functioning of the heart and circulatory system. During the action potential, Ca2+ ion influx via L-type Ca2+ channels triggers more Ca2+ release via ryanodine receptors in the adjacent sarcoplasmic reticulum. A variety of Ca2+ channels are also involved in the removal of this Ca2+ from the cytoplasm. Many of the proteins involved in both Ca2+ influx and efflux are located at invaginations of the ventricular myocytes, known as t-tubules including (reviewed in Orchard & Brette 2008). They make up over half the total cell membrane of atrioventricular myocytes (Pásek et al 2008) and form a network of transverse and longitudinal elements throughout the cell that ensures synchronous Ca2+ influx, contraction and rapid efflux. Thus the study of the these structures is very important in understanding how the action potential is linked to cardiac muscle contraction. For instance, we have shown that tonic phosphorylation by PKA- and CamKII-dependent phosphorylation of L-type Ca2+ channels, which are localised at t-tubules, maintains ICa (Chase et al 2010) while Ca2+ efflux from the sarcoplasmic reticulum via the Ca2+ ATPase occurs only at these tubules (Chase & Orchard 2011).
The AV node possesses intrinsic pacemaker activity but the mechanisms that underlie this crucial function are poorly understood. We have shown that isolated AVN cells display spontaneous Ca2+ transients and action potentials (Ridley et al 2008). Ca2+ transients could be blocked by thapsigargin and a reduction in extracellular [Na+], indicating the involvement of the Na+-Ca2+ exchanger (NCX) and the sarcoplasmic reticulum Ca2+ATPase (SERCA). In contrast, Ca2+ transients were stimulated by treatment with the β-adrenergic agonist isoprenaline. More recently we have demonstrated that inhibition of either the NCX or SERCA blocked both spontaneous Ca2+ transients and action potentials (Cheng et al 2010).
The regulation of ion channels is an obviously important area of research - ensuring that the complex sequence of channel opening and closing required to generate the cAP occurs correctly is critical to preventing arrhythmia. Phosphorylation-dephosphorylation cycles are a very common mechanism for controlling ion channel gating properties, but the precise effect they have can be misleading. Thus, it has long been established that the NCX can be phosphorylated by PKA and PKC, which can lead to increased NCX activity (e.g. Zhang et al 2001, Pabbathi et al 2002). However, we have recently shown that the apparent increase in NCX function in rabbit ventricular myocytes following β-adrenoceptor/PKA activation is likely to be due to the activation of a PKA-activated Cl- channel, most likely the CFTR channel (Barman et al 2011). These channels have recently been shown to be localised in large clusters in the sarcolemma (James et al 2010), although their role in cardiac function is as yet unclear.
The complex mechanisms by which phosphorylation of RyR2 by multiple protein kinases (including protein kinase A, calmodulin-dependent kinase II, and protein kinase C) can regulate the opening of the channel is another focus of research. Experiments have revealed how changes in these mechanisms can destabilise the normal gating behaviour of RyR2 channels (Carter et al 2011). This is particularly relevant for understanding heart disease. In heart failure, cardiomyocyte dysfunction is linked to changes in intracellular Ca2+ movements and alterations in the phosphorylation levels of many proteins including RyR2.
The roles of FKBP12 and FKBP12.6 (FK506-binding proteins) as regulators of RyR2 channels have been under investigation. Reduced FKBP12.6 binding to RyR2 has been implicated in mediating disturbances in Ca2+-homeostasis during heart failure but the underlying mechanisms were not understood. We have now shown that FKBP12.6 is a partial agonist with such a low ability to activate RyR2, that its main effect is to act as an antagonist of the closely related and much more abundant protein, FKBP12 (Galfré et al, 2012). We demonstrated that FKBP12 binds with extremely high affinity to RyR2, sensitising the channel to cytosolic Ca2+ and resulting in a marked increase in channel opening. Consistent with these results, physiological concentrations of FKBP12 increased Ca2+-wave frequency in cardiac cells, an effect that was antagonised by FKBP12.6. We therefore propose that FKBP12 and FKBP12.6 jointly regulate cardiac Ca2+ release and that the ratio of FKBP12.6:FKBP12 may contribute to the ‘leakiness’ of RyR2 channels. If this ratio is reduced in heart failure, the effects of FKBP12 may dominate, leading to an increased sensitivity of RyR2 channels to cytosolic Ca2+. This may result in elevated diastolic leak of Ca2+ and the arrhythmias characteristic of heart failure.