Repolarization Reserve in Cardiac Cells

Transcription

1 Journal of Medical and Biological Engineering, 26(3): Repolarization Reserve in Cardiac Cells Edward Carmeliet * Laboratory of Physiology, University Leuven, Leuven, B-3000 Belgium Received 14 June 2006; Accepted 8 July 2006 Abstract Repolarization reserve is a term used to indicate the existence of redundancy of repolarizing currents in cardiac cells. It provides safety and prevents cardiac arrhythmias due to excessive prolongation of the action potential duration (APD) and generation of early afterdepolarizations. The existence of repolarization reserve in cardiac cells and its modulation is the main topic of this short review. Under physiological conditions repolarization reserve is modulated by increase of frequency, sympathetic stimulation and the accompanying rise in [Ca 2+ ] i, [Na + ] i and [K + ] e. The analysis of the changes by frequency and activation of the sympathetic system reveals an increase in repolarization reserve. At elevated rates APD shortening is caused by enhancement of outward currents (I Kr, I Ks and I Na,K ), while inward currents are decreased (I Na, late and I CaL due to faster inactivation; peak I CaL may be increased by acceleration of recovery from inactivation). Under sympathetic stimulation not only sinus rate increases but specific effects occur at the ventricular level by activation of α- and β-receptors. Major effects occur via activation of β-receptors which result in an increase of I CaL, I Kr, I Ks and I Na,K, elevation of the plateau and further shortening of the APD. Pathological reduction of repolarization reserve with increased risk of deadly arrhythmias can be of hereditary (congenital LQTsyndromes) or of an acquired nature. One of the more frequent and dangerous forms of acquired LQT is caused by the use of medications. The drugs responsible belong to different groups but have the common effect of blocking K + currents. Since not all patients using these drugs show complications of arrhythmias attention should be given to the underlying risk factors, such as hypokalemia, hypomagnesemia, female sex, predisposing DNA polymorphisms, congestive heart failure, left ventricular hypertrophy. It is the aim of future research, especially in the case of hypertrophy and failure to unravel the remodeling mechanisms responsible for increased risk. In this way it will become possible to prevent excessive reduction of repolarization reserve and to preserve a normal repolarization. Keywords: Repolarization reserve, Frequency, Sympathetic stimulation, Ion concentrations, LQT, Remodeling Introduction Redundancy is a characteristic property of biological systems. It provides safety and prevents pathological disturbances. In cardiac electrophysiology the repolarization process of the action potential is guaranteed not by a single but by multiple currents. The contribution of these currents varies with cell type. Blocking of one current does not result in failure of repolarization. Compensation by other outward currents prevents dangerous arrhythmias and has been called repolarization reserve [1]. The action potential in nerve, skeletal muscle and cardiac cells is characterized by a rapid depolarization phase or upstroke which is caused by activation of the fast Na + conductance. Repolarization in cardiac cells is very different from that in nerve and skeletal muscle. In these latter cells repolarization immediately follows the depolarization phase and total membrane conductance is high. Repolarization in cardiac cells is characterized by the existence of a long plateau during which the rate of voltage change is very slow. The * Corresponding author: Edward Carmeliet Tel: ; edward.carmeliet@med.kuleuven.be return to the resting potential is delayed. The membrane conductance during the long plateau is about four times lower than during diastole [2]. It is caused by a dramatic fall in the K + conductance of the I K1 channel which is normally responsible for the negative membrane resting potential (Fig 1; see later inward rectification). The fall in outward K + current has the great advantage that K + loss during the long plateau is minimal. Energy waste via active transport is thus held at a safe lower limit. However, since net current as well as total current are small, any further important decrease of outward current may lead to excessive prolongation of the action potential duration (APD) and of the QT interval in the ECG. During recent years congenital as well as acquired forms of LQTsyndromes (LQTs) have been described (for review see[3]). When repolarization rate falls below 0.1V/s the probability for early afterdepolarizations (EADs) and extrasystoles rises[4]. Evolution to full-sized polymorphic tachycardia or torsade de pointes arrhythmia (TdP) is favoured by the existence of dispersion in the effective refractory period (ERP), which is caused by non-uniform prolongation of the APD over the wall of the ventricle. Fortunately this doom

2 98 J. Med. Biol. Eng., Vol. 26. No Figure1. Schematic representation of a cardiac action potential (top) and accompanying change in I K1 current (bottom). The I K1 current shows a dramatic fall during the plateau phase, due to block by intracellular Mg 2+ ions and positively charged polyamines. Unblock occurs during the final repolarization phase. Tracings are not actual recordings but drawings based on information provided by reference [4]. Table 1. Three phases of repolarization in cardiac cells Phase 1 or initial fast repolarization inactivation of I Na, activation of I to Phase 2 or plateau voltage-dependent currents inward currents: I CaL outward currents: I Kr, I Ks transporters: I NCX (in and out), I NaK (outward) Phase 3 or terminal repolarization : I K1 inward rectifier scenario does not occur frequently because of the existence of redundancy in the repolarization process. The existence of repolarization reserve in cardiac ventricular cells and its modulation is the main topic of this contribution. The normal repolarization process The repolarization in cardiac ventricular cells is generally subdivided in three phases, during which a number of inward and outward currents participate (for references see [5-6] (Table 1). Phase 1 or initial fast repolarization follows the upstroke of the action potential It is caused by inactivation of the fast inward Na + current (I Na ) and simultaneous activation of a transient K + outward current. The transient outward current is mainly carried by a voltage-activated current, I to (also called I to1 ) with fast activation followed by fast inactivation, but a Ca 2+ -activated Cl - current, I Ca.Cl (I to2 ), which rises in parallel with intracellular Figure 2. Schematic representation of the time course of the two delayed K+ currents, I Kr and I Ks (bottom) during the cardiac action potential (top). Note the late peaking of I Kr. Based on information in reference [4]. Ca 2+ concentration, can also contribute to the outward current. Phase 1 is variably expressed in different cardiac cells. It is very pronounced in Purkinje cells and M cells, large in subepicardial but small in subendocardial cells. It also varies with species: in guinea pig it is much less present than in human. The rate of voltage change is in the order of 10 V/s. Phase 2 or plateau follows phase 1 During the plateau the maximum repolarization rate is a thousand times smaller than during depolarization: 0.1 to 0.3 V/s compared to 200V/s to 500 V/s. The net current ( pa/pf) as well as the total conductance of the membrane are very small. The major cause for the small total conductance is the dramatic fall in I K1 conductance, the K + channel that is normally responsible for the resting potential. Different outward and inward currents contribute to the plateau repolarization. Outward currents Two voltage-dependent K+ currents are activated during the plateau, I Kr and I Ks, the rapid and slow delayed K+ currents respectively (Fig 2). Contrary to expectation for a rapidly activated current I Kr does not carry any large current early during the plateau but shows a peak only late at the end of phase 2 [7]. This is not due to the activation process as such: I Kr channels are activated early for depolarizations corresponding to the peak of the action potential (AP) (time constant of about 40 ms). Current through the I Kr channel however is small or absent as long as the membrane potential is positive to the zero level. The reason is inactivation, which in this channel is actually a faster process than activation [8]. Channels remain closed and only open when the membrane potential is brought back to around zero mv. In the current-voltage (I-V) relation such a behaviour is seen as fall of current (inward rectification) at positive membrane potentials. Following recovery from inactivation at more negative potentials the channels open and slowly deactivate. This succession of events is responsible for the transient nature of the I Kr current.

3 Repolarization Reserve in Cardiac Cells 99 The second outward current is I Ks or slow delayed K + current. In voltage clamp experiments using the action potential clamp method, the current has been shown to carry a slowly rising current over the whole plateau duration [9-11]. The total charge carried by I Ks is variable with species and conditions and is usually less than that of I Kr. In the absence of sympathetic stimulation, block of I Ks produces little APD prolongation in isolated rabbit, dog and human cardiac myocytes. The distribution of I Ks differs between cells: its expression in M cells is smaller than in other myocardial layers; in both subepicardial and subendocardial cells the contribution of I Ks is prominent. In this way I Ks determines to a large extent transmural dispersion of APD, the longest AP being observed in M cells [12]. The heterogeneity of I Ks expression in different cells also explains why block of the other delayed K + current, I Kr, affects M cells preferentially. Although I Ks block has normally only a small effect on APD, the effect of I Ks block becomes prominent when the AP is already prolonged. This is i) because the net outward current in a prolonged AP is smaller and any further decrease in outward current will have a larger proportional effect and ii) because more I Ks can be activated during a long AP (activation is time-dependent). The proportion of I Ks also enhances under sympathetic stimulation and block of the channels will become more efficient [10] (see section on sympathetic stimulation). Na +, K + -pump current (I Na,K ). Active Na +, K + -transport is electrogenic. In the range of membrane potentials corresponding to the action potential the pump current is outward. The amplitude is sensitive to Em, [Na + ] i and [K + ] e, with [Na + ] i as the more important modulator. This explains why pump rate and thus current is so sensitive to frequency of stimulation. The density of pump sites is larger in subepicardial than subendocardial cells, a characteristic distribution similar to that of I Ks and of interest for our understanding of the transmural potential gradient [13]. Inward currents Ca 2+ current (I CaL ). I CaL is activated early during the AP and carries a substantial charge into the cell: its mean density is about 5 times the net current during the repolarization process [4]. The current undergoes a voltage-dependent inactivation which is rather slow. A much faster inactivation is caused by the rise in free Ca 2+ ion concentration following influx of Ca 2+ ions from the extracellular medium and release from the sarcoplasmic reticulum (SR). Ca 2+ -dependent inactivation occurs via binding to calmodulin, which is prebound to the C-terminus of the channel. Part of the C-terminus then moves in the direction of the inner pore and blocks the channel [14-15]. When repolarization is very slow in the range of membrane potentials where activation and inactivation overlap, Ca 2+ channels can become reactivated: this extra inward current generates secondary depolarizations or EADs. Plateau Na + current. Most of the fast Na + current is inactivated early during phase 1 of the repolarization. During the plateau the Na + current however does not drop to zero. In a limited range of potentials where activation and inactivation overlap a small steady-state Na + current or window current can be recorded. Also a small component of slowly inactivating Na + current can be measured over a broad range of potentials in many types of cardiac cells of different species [16], including expressed human cardiac Na + channels [17]. The non-inactivating or slowly inactivating current also called late or persistent current is enhanced in the congenital LQT3 syndrome [18], in chronic ischaemia [19], hypertrophy and failure [20-21] (for review see [22]). Na +, Ca 2+ exchanger current (I NCX ). Transport of Ca 2+ ions through the NCX is responsible for maintaining intracellular Ca 2+ at a physiological low level (for review [23-24]). Since three Na + ions and one Ca 2+ ion are transported per cycle the transport is electrogenic. At rest the reversal potential for the exchanger is positive to the resting membrane potential: Ca 2+ is moved out of the cell, Na + is moved in and the current is inward. During the initial part of the AP the situation reverses: Ca 2+ moves in and the current becomes outward. Later during the plateau, when the cytoplasmic Ca 2+ concentration reaches its peak the reversal potential shifts positive to the membrane potential, causing Ca 2+ again to be moved out and producing inward current [25]. The I NCX thus will mainly carry inward current. In situations of Ca 2+ overload I NCX may be responsible for critically reducing the rate of repolarization late during the plateau, allowing Ca 2+ channels to be reactivated and generate EADs [26]. Phase 3 or final repolarization: I K1. In the Introduction we mentioned that a fall in I K1 was the main phenomenon responsible for the long APD. The rate of repolarization during the plateau is dependent on the interplay between outward currents through I Kr, I Ks, I Na,K and to a minor extent through I NCX, and inward current through I CaL and I NCX. When the membrane potential approaches the level of 50 mv repolarization speeds up reaching values of 0.5 to 5 V/s (variable with type of cell and species). The main mechanism responsible for this faster repolarization is outward current through I K1 [27]. The inward rectifier current is indeed a major determinant of the final repolarization and carries a substantial charge (about 4 times that of I Kr at the end of the plateau). This increase in outward current is not due to a voltage-dependent activation but to an unblocking of the channel. At voltages positive to 50 mv the channel is blocked by Mg 2+ and polyamines which enter the channel from the intracellular side in a potential-dependent way [28]. At more negative levels the channel is unblocked and outward current increases. This greater outward current at more negative potentials generates a negative resistance and the I-V relation is characterized by inward rectification [29]. The system forms a positive feedback (Fig 3): the greater the hyperpolarization, the less block by Mg 2+ or polyamines and thus the greater the outward current which in turn causes more hyperpolarization. Repolarization is thus a regenerative phenomenon. During phase 3 the regenerative process is graded and, once started, progresses to completion; the instantaneous I-V relation contains a region of negative slope but is outward over the whole voltage region. Earlier during the plateau regenerative repolarization can be induced by hyperpolarizing impulses and shows a threshold similarly to the Na + conductance system

4 100 J. Med. Biol. Eng., Vol. 26. No Figure 3. Steady-state I-V relation of the I K1 current in cardiac cells (top). Note the strong inward rectification, with a region of negative resistance between 65 mv and 20 mv. During phase 3 of the cardiac action potential the existence of negative resistance in the I-V relation generates a positive feedback (bottom) and is responsible for the regenerative nature of the final repolarization. Schematic drawing based on reference [27]. during the depolarization [30-31]. The existence of a threshold means not only that there is a negative resistance but that the instantaneous I-V crosses the voltage axis with outward current negative and inward current positive to the threshold voltage. Physiological modulation of repolarization reserve Repolarization reserve is variable: net current and total conductance of the membrane during the repolarization process are not fixed. Under physiological conditions repolarization reserve is modulated by increase in frequency of stimulation, sympathetic stimulation and the accompanying increase in [Ca 2+ ] i, [Na + ] i and [K + ] e. All these processes are interrelated as shown schematically in Fig 4. From an electrophysiological point of view sympathetic stimulation has two important effects on the heart: i) it increases substantially the spontaneous sinus node rate, and ii) at the level of atrial and ventricular cells it modulates a number of ionic currents and transporters. The relative amplitude of both effects depends on a dynamic equilibrium between activity of the right and left stellate ganglion. The sinus rate is selectively innervated by postganglionic fibers of the right stellate ganglion, while most of the left ventricular myocardium is governed by the left stellate ganglion. We will first discuss the effect of rate as such. Effect of rate. The higher the frequency, the greater the shortening of the APD in steady state (for review [22]). Initial changes for a sudden increase in rate differ between species and type of preparation. In human ventricle an important initial shortening is followed by a slow and gradual shortening of APD, which develops over a time course of tens of seconds to a few minutes. The underlying processes responsible for the APD shortening demonstrate an increase of repolarization reserve. Inward currents decrease whereas outward currents increase: at higher rates the safety factor for repolarization is enhanced. Figure 4. Interplay between sympathetic stimulation, heart rate, change of [Na + ] i, [K + ] e and [Ca 2+ ] i in modulating repolarization reserve. Most of the currents and transports indicated in this scheme are enhanced; negative changes are explicitly mentioned. Sinus rate is augmented via β-receptor stimulation and activation of a number of ionic currents. Increase in sinus rate decreases I Na,late and enhances I Ks and I Kr because of the specific kinetics of these currents. I Na,late shows slow recovery from inactivation. For I Ks activation is accelerated ; at high rates in some species I Ks tails may also show summation. For I Kr the increase is the consequence of the change in AP shape and the accompanying changes in activation-inactivation. Slower changes in ionic currents with increase in sinus rate are secondary to increase of [Na + ] i, [Ca 2+ ] i and [K + ] e. The increase in sinus node rate is normally caused by activation of the right stellate ganglion of the sympathetic system. Stimulation of the left stellate ganglion causes release of norepinephrine at the level of left ventricular cells with activation of α- and β-receptors, followed by activation of PKC and PKA and subsequent phosphorylation of a number of ionic channels and transporters (SERCA or sarcoplasmic reticulum Ca 2+ -ATP-ase and I Na,K or Na +,K + -ATP-ase). Later epinephrine, set free from the adrenal medulla, will also reach the heart. In general, currents are enhanced, except in the case of I Na,late. I NCX is modulated by changes in [Na + ] i, [Ca 2+ ] i and PKC-dependent phosphorylation; depending on the reversal potential the current can be inward or outward. For further information see text and [3, 61, 22]. Inward currents during the plateau are decreased. At higher rate of stimulation, I CaL declines faster. The mechanism is amplified Ca 2+ -dependent inactivation consequent to the enhanced release of Ca 2+ ions from the SR. The peak current however, may be increased by Ca 2+ -dependent facilitation. Recently Ca 2+ -dependent activation of endogenous Calmodulin kinase II has been shown to play an important role in this facilitating mechanism by accelerating recovery from inactivation [32]. The late Na + current or slowly inactivating Na + current is also reduced. Two mechanisms play a role. i) Recovery from inactivation of the late Na + current, in contrast to that of the fast I Na is characterized by a slow time course [33]. At higher rates of stimulation with shorter diastole, recovery from inactivation thus remains incomplete and causes a fall in

5 Repolarization Reserve in Cardiac Cells 101 current. ii) A second reason for a decline in current is a reduction in maximum conductance caused by PKC-dependent phosphorylation of the channel as a consequence of increase in intracellular free Ca 2+ concentration at higher rates [34]. In contrast to inward currents, outward currents are increased at higher stimulation rates. Summation of tail currents at high rates has been described for I Ks in guinea pig ventricle, but this does not seem a general mechanism. In many species deactivation is too fast to allow important summation at short diastolic intervals [7, 35-36]. The kinetics of I Ks activation during the AP on the other hand are accelerated and outward current reaches higher levels when stimulation rate increases [7]. The change in kinetics and the increase in conductance have been explained in the following way. Opening of the channels is not a single reaction from closed to open but requires passage through a number of intermediate non-conducting states. At the end of an AP not all channels have reached the open state but a number have moved near to the open state. During diastole the channels follow the reverse reaction but not all return to the rested state. Some remain at an intermediate state from which activation during the next AP is more readily turned on (for a modeling study see [37]). This creates an available reserve of channels that are ready to open on demand. A repolarization reserve is generated in the channel itself. A second mechanism for a rise in amplitude of I Ks with rate of stimulation is increase in conductance following phosphorylation of the channel by PKC. This enzyme in turn is activated by the rise in [Ca 2+ ] i at elevated rates of stimulation [38]. Phosphorylation however is not the only mechanism by which [Ca 2+ ] i modulates I Ks. A phosphorylation-independent but calmodulin-dependent stimulation has been suggested by Nitta et al.[39]. Recently two groups [40-41] have demonstrated the existence a direct binding of Ca 2+ to the channel complex via calmodulin. This binding facilitates channel assembly, prevents inactivation, shifts the activation to more negative potentials and thus mediates a considerable Ca 2+ -sensitive increase of the I Ks current. Modulation of I Kr seems less developed. The existence of a direct effect of rate, i.e. summation and/or faster activation kinetics, is more controversial. No effect has been found in the guinea pig [35] or in the dog [36]. According to Gintant [36] this is due to the rapid activation of I Kr, which is complete within 150 ms or the duration of a single AP. In the guinea pig Rochetti et al [7] explained the increase in I Kr level at higher rates of stimulation by taking into account the accompanying change in shape of the AP. The dependency of I Kr on AP shape, according to the authors, can be justified by its known kinetic properties of activation and inactivation. This is a remarkable result because the system forms a positive feedback system: the higher the rate of repolarization the greater the activation level of I Kr. As for I Ks, I Kr has been shown to be phosphorylated by a Ca 2+ -dependent PKC. The result is a marked decrease in inward rectification which means enhancement of current at positive potentials [42]. As a final comment one should stress the fact that not only [Ca 2+ ] i but also [Na + ] i and [K + ] e rise at higher frequency (Fig 4). The rise in [Na + ] i will stimulate the Na +,K + -pump and generate extra outward current during the whole plateau favouring repolarization (review see [22]). [K + ] e increase also stimulates the pump, and exerts moreover an enhanced effect on I Kr [43] and I K1 [44]. Sympathetic stimulation. The effect of rate has been discussed in the previous section: an increase in rate shortens the APD, it enhances repolarization reserve. We will now concentrate on changes in currents and transporters by sympathetic stimulation which are due to interaction of norepinephrine and epinephrine with specific receptors in ventricular cells. Norepinephrine, released from the nerve endings, and epinephrine, set free from the adrenal medulla, bind to α- and β-receptors in the target cell. In heart the major effects occur via β-receptor stimulation. Three pathways are involved: direct activation of G-proteins, binding of camp and, the most important, camp-mediated activation of PKA and phosphorylation of the target. β-receptor activation causes an increase in I CaL, I Ks and I Na,K and results in elevation and shortening of the AP plateau. Ca 2+ current. I CaL is enlarged by β-receptor stimulation via PKA-dependent phosphorylation. The effect is an increase in conductance (recruitment of more channels) and acceleration of activation [45]. The increase in I CaL would normally prolong the APD in ventricular cells and this effectively occurs at low concentrations of norepinephrine where stimulation of I CaL prevails [46]. A larger I CaL at higher concentrations of norepinephrine however, also means a more positive plateau. This larger depolarization allows for greater activation of I Kr and I Ks. Since outward currents at these concentrations are positively modulated by direct β-receptor stimulation (I Ks see below) and rise of [Ca 2+ ] i (I Ks and I Kr ) the final effect under normal physiological conditions is a higher plateau but shorter duration. Delayed K + currents. As noted in the previous discussion on rate effects, both I Kr and I Ks are enhanced by Ca 2+ -dependent PKC stimulation. In the presence of sympathetic stimulation the rise in [Ca 2+ ] i will be amplified by an enhanced Ca 2+ influx through I CaL and larger storage in the SR by a stimulated Ca 2+ -ATPase The I Ks channel furthermore, is specifically stimulated by Ca-calmodulin-dependent and PKA-dependent phosphorylation. It has become evident that the I Ks channel, composed of KCNQ1 and KCNE1 proteins, forms a macromolecular complex with calmodulin and with PKA and β-receptor signaling molecules [47][40-41]. Modulation of I Ks by Ca-calmodulin has been discussed in the section on rate. Phosphorylation by PKA moreover, shifts kinetics and increases conductance and causes a faster and greater outward current during the AP [48]. The changes in both delayed K + currents are primordial in causing shortening of the APD under sympathetic stimulation. The absence of one of the delayed K + currents in congenital LQT syndromes explains why catecholamines may induce QT prolongation under those conditions [49]. The effect on I CaL then prevails. α-receptor increases the activity of the Na +,K + pump- ATPase and of the Na +, Ca exchanger via activation of lipases

6 102 J. Med. Biol. Eng., Vol. 26. No Table 2. LQT syndromes and secondarily of PKC [50]. At the channel level α-receptor stimulation inhibits I to, I K1 and late I Na Some of these actions result in shortening others in prolongation of APD. Under normal physiological conditions the effect of β-receptor activation on heart is prevalent over the effect of α-receptor activation. It may thus be concluded that repolarization reserve under activation of the sympathetic system under normal physiological conditions is substantially enhanced. Pathological changes in repolarization reserve Congenital LQT syndromes (LQTs) (for review see [3, 51]) A great number of mutations of channel or transporter genes resulting in dramatic reduction of repolarization reserve have been described during recent years. Most of these mutations cause reduction of repolarizing currents ( I Kr, I Ks, I K1, and I Na,K ), only one type concerns a gain of function and causes an increase of the late inward Na + current (see table 2). Loss of function is due to a deficient trafficking of channel molecules to the cell membrane, to changes in kinetics, conductance or selectivity of the channel. Gain of function in the case of I Na, is caused by slowing of inactivation. They all result in congenital LQTs characterized by life-threatening arrhythmias and often sudden death. Fortunately these mutations are relatively rare. Loss of I Ks channel function is seen in LQT1 and LQT5 due to mutations in KCNQ1 and KCNE1 gene, loss of I Kr is characteristic for LQT2 and LQT6 due to mutations in KCNH2 and KCNE2, loss of I K1 in LQT7 is due to mutation in KCNJ2. A gain of function in the late Na + channels is characteristic for LQT3, which is caused by mutations in SCN5A. The LQT4 is caused by deficient functioning of an anchoring protein, ankyrin-b, normally responsible for membrane targeting of the Na +,K + pump-atpase and NCX protein. The result is aberrant intracellular Ca 2+ regulation. In all these mutations the QT interval or cellular APD is increased to a large extent such that EADs may be generated. The majority of serious arrhythmias in LQT1, LQT5 and LQT2 and LQT6 syndromes occur during exercise or emotional stress, when an increase in sympathetic activity is expected [52]. This may seem strange since sympathetic stimulation has been shown to enhance repolarization reserve in normal physiological conditions via increase in outward and decrease in inward currents. The reason for this negative effect is that excessive stimulation of the sympathetic system on the background of reduced outward currents may increase I CaL to such extent that EADs are generated. The reduction of outward current caused by the mutation, can be due to deficient channels or in the case of I Ks to uncoupling of the channel from the PKA system [47]. As mentioned previously the I Ks channel forms a macromolecular complex together with β-signaling molecules. In some mutations linked to LQT1 and LQT5 sympathetic stimulation is disrupted in such a way that I Ks phophorylation is deficient. I Ks is uncoupled from PKA modulation, whereas I CaL modulation is still normal. The enhanced I CaL may then lead to excessive prolongation of APD and EADs. Experimentally, β-receptor stimulation has been shown to prolong QT in LQT1 and LQT2 patients and to increase transmural dispersion in LQT1 and LQT2 animal models [53, 49, 54]. Excessive prolongation of the AP and existence of a high grade of dispersion are pivotal mechanisms in the genesis of arrhythmias. The increase in dispersion is caused by a selective increase of APD in M cells while APD is shortened in subepicardial and subendocardial cells. Due to the mutations, I Ks and I Kr are less expressed especially in M cells, and the APD under β-receptor stimulation will be prolonged. In subepicardial and subendocardial cells on the other hand, shortening of APD is caused by enhancement of I Ks and I Kr which are sufficiently expressed and represent a substantial repolarization reserve against I CaL. In LQT3, arrhythmias are generated preferentially during rest or sleep, when cardiac frequency is very low. Excessive APD prolongation at long cycle lengths has been confirmed in the transgenic KPQ mouse model [18]. EADs may develop under those circumstances and are responsible for the initiation of arrhythmia. At higher rates, summated inactivation of the late current [33] together with larger and faster activation of I Ks will act in the opposite way and tend to normalize the APD as a consequence of enhanced repolarization reserve. The antiarrhythmic effect of isoproterenol can be explained by the net increase of repolarizing current (K + currents and Na +,K + pump current) by β-receptor stimulation and possibly by a reduction of late I Na as a result of PKC-dependent phosphorylation induced by increase of intracellular Ca 2+. β-blockers are the mainstay therapy for LQT1 and LQT2 patients. This is reasonable since most of the arrhythmias in these patients occur in the presence of sympathetic overdrive. For LQT3 patients however the usefulness of β-receptor therapy has been questioned [55]. During rest or sleep when most cardiac events occur, β-receptor antagonists may further slow heart rate and promote arrhythmias. At elevated rates on the other hand, β-receptor agonism (isoproterenol) exerts antiarrhythmic activity in the KPQ transgenic mouse model [18]. In LQT3 patients and cellular models, β-receptor stimulation antagonizes LQT and causes shortening of the APD independent of the rate. The use of β-blockers thus remains questionable.

7 Repolarization Reserve in Cardiac Cells 103 Acquired LQT syndrome. The LQT syndrome is not necessarily of hereditary origin but can also be of an acquired nature [1]. One of the more frequent and dangerous forms of acquired LQT is caused by the use of medications. The drugs responsible belong to different groups but have the common effect of blocking K + currents. Among the antiarrhythmics, drugs of class I and III should be mentioned: procainamide, quinidine, disopyramide, sotalol, dofetilide, ibutilide. Others belong to the antihistamines e.g. terfenadine, astemizole; antimicrobials e.g. erythromycine, ketoconazole; gastrointestinal drugs e.g. cisapride, and psychotropic drugs e.g. haloperidol. Chemically they all have at least one aromatic ring, they block I Kr and are trapped when the channel closes [56]. During recent years it has become evident that I Ks under conditions of I Kr block can compensate for the loss of I Kr [11]. This is due to the longer and more positive plateau, allowing more channels to open and more channels to move close to the open state such that they readily open during the next AP. Not all patients using these drugs show complications of TdP arrhythmias. It is thus important to delineate the risk factors that amplify the danger of blocking certain repolarizing current or reducing repolarization reserve. Among these factors one should mention hypokalemia, hypomagnesemia, congestive heart failure, left ventricular hypertrophy, female sex [57] and predisposing DNA polymorphisms [1]. The hypertrophic and failing heart undergoes a number of remodeling modifications in the myocytes which are predisposing to arrhythmias [58]. Ventricular ectopy and arrhythmias are a frequent complication which may result in sudden death. The mechanisms involved are enhanced tendency to triggered activity on one hand and favourable conditions for reentry on the other hand. The fall in different K + currents and of I Na,K shift the background in the inward direction and slow repolarization. The occurrence of EADs is favoured by the prolongation of the APD especially at low frequencies of stimulation. Increased I NCX again plays a conditioning role and facilitates reactivation of I CaL [59]. It is the aim of future research to unravel the underlying remodeling mechanisms. In this way it will become possible to prevent or reverse remodeling and avoid LQT arrhythmias. Conclusions Redundancy in repolarizing currents, called repolarization reserve, guarantees a normal repolarization process in cardiac cells. Increase in rate and sympathetic stimulation under physiological conditions enhances repolarization reserve. Pathological reduction of repolarization reserve which increases the risk of deadly arrhythmias can be of hereditary (congenital LQT syndromes) or acquired nature. By way of conclusion, the following aspects should be stressed. 1. The effects of sympathetic stimulation at the level of ionic channels are multiple and interrelated. Changes in ionic currents are caused via modulation of kinetics and conductance, trafficking and assembly of the channel, PKA- and PKC-dependent phosphorylation. An important component is the increase in [Ca 2+ ] i which modulates a number of currents via PKC-dependent phosphorylation, stimulates I Ks via Ca 2+ -CaM-binding, and activates I NCX acting as a substrate. The rise in [Ca 2+ ] i feeds back on I CaL itself: in a negative way by accelerating inactivation (Ca 2+ -CaM binding) and in a positive way by accelerating recovery from inactivation (Ca 2+ -CaMkinase II). Under physiological conditions the total result is elevation of the plateau level and shortening of the APD. 2. Under conditions of pathological prolongation of the APD (LQT syndrome), activation of the sympathetic system causes dual effects. When delayed K + currents are deficient, the amplified I CaL will act as an arrhythmogenic by further prolonging the APD and favouring EADs. It explains why the majority of arrhythmias occur during exercise or stress in LQT1, LQT2, LQT4 and LQT6. However, when APD prolongation is caused by a gain in I Na,late, i.e. in LQT3, arrhythmias are generated preferentially during rest of sleep, when frequency is low. In this case, β-receptor stimulation has been shown to be antiarrhythmic, because of the fall in I Na,late and the concomitant increase in I Ks and I Na,K currents. 3. Excessive increase in repolarization reserve has not been discussed in the present review. It may occur in atria and favour the transition from acute to chronic atrial fibrillation. Remodeling as a consequence of high rate stimulation causes a drastic reduction in the expression of Ca 2+ channels and induces a stable shortening of the APD with enhanced probability of reentry arrhythmias [60]. 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