CARDIAC BACKGROUND

Figure 1. Prolongation of the Ventricular Action Potential (ECG vs. Action Potential Duration). Figure 2.Ventricular Action Potential (Action Potential Currents Selected), some currents left out for clarity.

The Cardiac Action Potential

Surface recordings made during the electrocardiogram (ECG) reflect the electrophysiological events occurring during impulse generation and conduction in the heart.  Each heartbeat starts as an electrical excitation generated in the sinoatrial node and is rapidly conducted throughout the atria.  On surface ECG measurements, the ‘P’ wave represents the combined electrical activity of action potential (AP) depolarisation in the atria (Figure 1).  Impulse conduction to the ventricles occurs though the atrioventricular node and is transmitted rapidly across both ventricles via the His-Purkinje system, by virtue of tight electrical coupling between ventricular cells.  The QRS complex of the ECG corresponds to the AP depolarisation as it occurs in the ventricles (masking the electrical activity associated with AP repolarisation in the atria), while the T wave is associated with ventricular repolarisation (Figure 1).  Thus the QT interval of an ECG represents the duration of the ventricular AP, plus the time associated with transmission across the myocardium (Belardinelli et al., 2003).  It therefore follows that a prolongation of the QT interval corresponds to a prolongation of the ventricular action potential (Figure 1).  In humans the ventricular AP is typically 200-300 milliseconds (ms) in duration.  Prolongation of a heart-rate corrected (QTc) interval[1] in excess of ~440 ms for men or ~460 ms for women has for sometime been associated with an increased cardiovascular risk (Schouten et al., 1991).

The AP of all ventricular cells shares many ionic and electrical similarities, although variation exists between species, region and function (Haverkamp et al., 2000).  The typical mid-myocardial ventricular AP is shown schematically (Figure 2).  Initially the cell is polarised near to the electrochemical potential for potassium ions (EK) because of high K+ conductance at rest.  A rapid depolarisation, mediated by a Na+ current, and not blocked by TTX in many species, is followed by a brief partial repolarization mediated by a transient outward current (Ito), consisting of two components, one a Cl- current, and the other by a K+ current.  Ito is the principal repolarizing current of ventricular AP in mice and rats, which have very fast heart rates. However, in humans, and other species used for studying cardiac cellular electrophysiology such as dogs, guinea pigs, rabbits, swine and ferrets, Ito is not the principal repolarising current of the ventricles (Crumb and Cavero, 1999).  The plateau phase of the action potential then follows; this is a dynamic equilibrium between the movement of Ca2+ into and K+ out of the cell.  As K+ efflux exceeds Ca2+ influx, the cell membrane slowly repolarises.  This repolarisation reduces the Ca2+ current which, augmented by an increase in conductance of some K+ channels, results in a positive feedback loop of increased repolarisation.  In the final stage of the AP, the rapid repolarisation phase, the Ca2+ current is comparatively small, and the augmenting K+ current rapidly induces complete repolarization, thereby restoring the original resting potential of the myocyte (Figure 2).

The rate of K+ efflux, and by extension the rate of repolarisation, is determined by the density and gating properties of different K+ channels.  Distinct K+ channels feature in the initiation and completion of AP repolarisation.  The delayed rectifier current (IK) is particularly important in regulating the duration of the AP plateau and comprises rapid (IKr) and slow (IKs) components (Figure 2).  Composite IK develops progressively during the plateau phase, opposing the inward currents underlying plateau depolarisation.  As the net balance of outward current exceeds inward current, repolarisation occurs.  At a cellular level, iatrogenic slowing of AP repolarisation can lead to the generation of spontaneous depolarisations on the falling phase of the AP plateau.  Unlike the AP itself, these events, termed ‘Early After Depolarisations’ or ‘EADs’ are not synchronised with an excitatory stimulus, and so can give rise to asynchronous tissue excitation and, thereby, arrhythmogenesis. 
Under physiological conditions the AP progresses through the phases of depolarisation, plateau phase and repolarisation over the course of 200-300 ms; however, in clinical conditions such as long QT syndrome (LQTS), repolarisation is delayed and the AP duration prolonged.  Electrophysiologically, this delay in repolarisation implies a deviation from the usual balance of currents across the myocyte membrane responsible for normal repolarisation.  Thus, prolongation of the AP could theoretically arise from an increase in inward (depolarising) current or with a reduction in outward (repolarising) current, both of which exist in different populations of LQTS patients.

Familial and Acquired Long QT Syndrome

Long QT syndrome (LQTS) exemplifies one of the best understood cardiac disorders.  “Long QT Syndrome” was originally coined by Jervell and Lange-Nielsen in 1957 who observed an association between prolongation of the QT interval of the ECG with syncope and sudden death. These symptoms are now intrinsically linked with Torsades de Pointes (TdP; Dessertenne, 1966) a distinctive polymorphic ventricular tachycardia (with concomitant QTc prolongation), characterised by continuously alternating QRS complexes, which can degenerate into ventricular fibrillation, ultimately leading to sudden death in most untreated patients.  Familial LQTS is the inherited form of this cardiac disorder, which affects young, otherwise healthy individuals (Ward, 1964; Romano, 1965).  Most LQTS gene carriers manifest with prolongation of the QT interval on an ECG, a sign of abnormal, slowed, cardiac repolarization (Vincent et al., 1992).  Familial LQTS is characterised by early symptom onset (mean age of 24 in the international registry, Moss et al., 1985), frequent, recurrent, non-sustained tachyarrhythmia’s, and a labile, prolonged QT interval on the ECG.  In a large prospective study Moss et al., (1991) reported that patients diagnosed as having LQTS had a higher frequency of pre-enrolment syncope (fainting/collapse) or cardiac arrest with resuscitation (80%), a resting heart rate less than 60 beats/min (31%), a history of ventricular tachyarrhythmia (47%), and a higher rate of congenital deafness (7%) than unaffected family members.  The arrhythmogenic syncope is often associated with acute physical, emotional, or auditory arousal, and these syncopal episodes are due to TdP.  Indeed, literature relating to the variant of the disorder associated with congenital deafness pre-dates the development of the ECG.  Meissner (1856) described a deaf girl who collapsed and died while being admonished at school.  The child had two brothers who also died suddenly after violent fright or rage.  In 1957 the first complete description of the disorder, including electrocardiographic information, was written by Jervell and Lange-Nielsen (1957) who described four deaf children with QT prolongation, three of whom died suddenly.  Thereafter, reports of LQTS without deafness, which were clearly distinct from the recessively inherited Jervell and Lange-Nielsen syndrome, appeared (Ward, 1964).  Descriptions of families involving multiple generations of LQTS sufferers were reported around the same time (Barlow et al., 1964; Romano, 1965; Garza et al. 1970), pointing to a higher incidence than originally thought.

Insight into the electrophysiological causes underlying LQTS has come from genetic studies, with linkage analysis now showing that of the seven genes associated with familial LQTS, six are ion channel subunits expressed in the heart (Table 1).  The first of these genes to be associated with LQTS was HERG (Human Ether-a-go-go Related Gene), a voltage-gated potassium channel alpha subunit that mediates the rapid component of the delayed rectifier potassium current, IKr (Curran et al.; 1995 Sanguinetti et al., 1995).  In addition to HERG (also known as KCNH2 or Kv11.1), others channels including a Na+ channel called SCN5A (Wang et al., 1995), a previously unidentified K+ channel called KvLQT1 (KCNQ1; Barhanin et al., 1996; Sanguinetti et al., 1996), and two putative K+ channel beta subunits, KCNE1 (mink; Splawski et al., 1997) and KCNE2 (hMiRP1; Abbott et al., 1999) have been identified (Table 1).  Most cases of familial LQTS are caused by defects in expression or regulation of ion channels controlling electrical activity in ventricular cells.  Even although the prevalence of familial LQTS is less than 1/100,000 (Puddu et al., 2001), in excess of 177 distinct mutations have now been found in LQTS patients (Walker et al., 2003).

When HERG was first demonstrated to be a K+ channel responsible for chromosome-7-associated (LQT2) familial LQTS (Curran et al., 1995), it was also suggested that pharmacological inhibition of HERG was a possible mechanism for acquired LQTS (Sanguinetti et al., 1995); acquired LQTS affects a much older patient group and is associated with administration of drugs that produce QT prolongation.  The subsequent finding that mutations in the gene encoding IKs were involved in a different form of familial LQTS (LQT1; Wang et al., 1996) raised the possibility that pharmacological depression of IKs might also produce, or contribute to, acquired LQTS.  However, while the role of IKs in human cardiac AP repolarisation has been questioned (Crumb and Cavero, 1999), it cannot yet be excluded (Redfern et al., 2003).  However, as discussed below (see ICH S7B; Redfern et al., 2003), HERG/IKr has become intrinsically associated with acquired LQTS and by analogy TdP (see ICH S7B; Redfern et al., 2003).

Human Ether-a-go-go Related Gene (HERG)

HERG was originally cloned by homology screening from a human hippocampal library (Warmke and Ganetzky, 1994).  In other species, transcripts are expressed strongly in brain, slightly less so in heart, testis and lung, with much lower expression found in skeletal muscle, adrenal gland, and thymus (Wymore et al., 1997).  The putative structure of the HERG channel (1159 amino acids), reveals HERG to be similar in many respects to other members of the shaker-type voltage-gated K+ channel families (Warmke and Ganetzky, 1994).  These K+ channels are made up of four subunits (Mackinnon, 1991), each of which has six a-helical transmembrane domains and a looping “pore region” (Papazian et al., 1987).  The transmembrane domains are functionally organised such that S5 and S6 and the looping pore region contribute to the pore, and the S4 region includes regularly spaced charged amino acids which function as the voltage sensor (Isacoff et al., 1990).  The HERG channel has N- and C- termini which lie intracellularily, with the former functionally involved in channel deactivation (Schonherr and Heinemann, 1996).  X-ray crystallography has shown the structure of the N-terminus to be related to a PAS regulatory domain (Morais-Cabral et al., 1998).

As discussed above, IKr encoded by HERG, is one of the major currents responsible for repolarisation of the cardiac myocyte ventricular AP (Sanguinetti and Keating, 1997).  HERG and IKr (Sanguinetti et al., 1995) are pharmacologically similar, being blocked by methanesulphonanilide class III antiarrhythmics, such as E-4031, MK-499 and dofetilide, which are archetypal high affinity HERG-blockers (Sanguinetti and Keating, 1997).  The unique kinetics of the HERG channel (inactivation faster than activation) means that relatively little current flows at the peak of the AP overshoot, because channels are rendered non-conducting by rapid inactivation (Zhou et al., 1998; Hancox et al., 1998, Pflügers Arch, 436(6):843-53.).  This means that during a ventricular AP, the channel current increases greatly during the repolarisation phase as inactivation is removed (Figure 2).  The inhibition of IKr leads to excessive lengthening of action potentials, which can induce EADs (leading to ectopic beats), and along with increased dispersion of ventricular repolarisation, probably underlies the cellular mechanism of TdP (Sanguinetti and Keating, 1997; Belardinelli et al., 2003).  The link between HERG/IKr, acquired QT interval prolongation and TdP has become one of the foremost issues in drug discovery and development (Fermini and Fossa, 2003).  With ever increasing regulatory scrutiny, any association of new chemical entities (NCEs) with these issues could adversely effect subsequent drug development (Crumb and Cavero, 1999).  The need to identify high throughput screens that can be used early in pre-clinical drug development to assess the potential for prolonged ventricular repolarisation is paramount.  Below we discuss the current regulatory position, and the development and use of new and old assays that may directly or indirectly detect compounds with arrhythmogenic potential at the pre-clinical stage.

[1]QTc is defined as the QT interval corrected for heart rate.  Several methods (> 30), for calculating QTc exist, although the most common is Bazzett.

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