**4. Mechanism 2: Triggered afterdepolarization**

The first is afterdepolarization or triggered activity. This ectopic event (a) arises within stressed or failing atrial (*or ventricular*) myocytes, (b) appears to require specific changes in intracellular signaling and post-translational protein modification including phosphoryla‐ tion, (c) is hypothesized to depend on changes in intracellular calcium homeostasis, and (d) needs a preceding action potential as a triggering event.

the turn of the twentieth century, the identification of impulse reentry in jellyfish, and its ascendance as a facile, malleable explanation for arrhythmia caused the focal view to fall in‐ to disfavor. By the middle of the twentieth century only few proponents supported it, in particular investigators like Rothberger, Scherf, and Kisch [16,26,27]. They continued to present data which showed that focal (*or cellular*) sites of spontaneous depolarization could provoke arrhythmia just as well as impulse reentry. From the 1920s through the 1950s Scherf repeatedly reported that focal administration of toxins like aconitine or alkaloids like vera‐ tradine incite cardiac rhythm disturbances that mimic atrial (*or ventricular*) fibrillation and atrial flutter. It is important to note that these pharmacological agents initiate arrhythmia by modifying sodium channel gating properties to disrupt this gatekeeper of the action poten‐ tial. Jervell and Lange-Nielsen published a groundbreaking report in 1957 [28] which first documented the long QT syndrome and laid the foundation for research on the genetic basis for arrhythmia. Dessertenne [29] and others greatly developed the appreciation that genetic mutation can alter the biophysical properties of voltage-dependent sodium and potassium channels in a manner analogous to the pharmacological approach of Scherf. Consequently, in addition to changes in the gross electrical properties of heart muscle proposed to underlie wavebreak and impulse reentry, pharmacological or genetic modification of ion channels came to be accepted as potential sources of clinical arrhythmia. But this toxin and genetic view have at least three critical limitations when used as evidence to support a cell-based

Toxins and alkaloids modify the biophysical properties of the sodium channel to provoke arrhythmic activity. These changes in channel properties at the site of toxin administration may provoke conditions that favor impulse reentry. Thus these pharmacological approaches

Mutations of voltage-dependent ion channels also might create conditions for functional or anatomic impulse reentry. Indeed reentry is invoked to explain genetically-linked arrhyth‐

Even if toxin-induced arrhythmia were purely a focal event, this approach to induce ar‐ rhythmia does not identify the cellular process which might alter the biophysical properties of the sodium or other voltage-dependent ion channels to recapitulate the arrhythmogenic

The development of a robust focal explanation for arrhythmia requires the identification of cellular mechanisms that destabilize quiescent atrium (*or ventricle*) to produce sporadic, ta‐ chycardic or fibrillatory ectopic electrical activity. There are two mechanisms now accepted

The first is afterdepolarization or triggered activity. This ectopic event (a) arises within stressed or failing atrial (*or ventricular*) myocytes, (b) appears to require specific changes in

might incite arrhythmia in a reentrant manner analogous to faradic sources.

focal hypothesis of arrhythmia

84 Atrial Fibrillation - Mechanisms and Treatment

mia including the long QT syndromes [30].

to generate such abnormal electrical impulses.

**4. Mechanism 2: Triggered afterdepolarization**

effects of aconitine or veratradine.

The groundbreaking work of Arvanataki in 1939 [31] provided the initial evidence for after‐ depolarization. This series of papers demonstrated that spontaneous electrical activity oc‐ curred in a wide range of excitable cells including snail muscle when these preparations were stimulated at extremely rapid rates and the pacing stimulus then was abruptly stop‐ ped. Studies reported by Bozler in 1943 [32] expanded on this breakthrough work, demon‐ strating that cardiac muscle also can afterdepolarize. The two types of afterdepolarization are designated as early or delayed events.

Early afterdepolarization occurs either during the Phase II plateau or during Phase III repo‐ larization of a prolonged action potential. Increased late sodium current [33] or decreased potassium channel activity, lowered 'repolarization reserve' [34], may prolong the duration of the action potential. Numerous studies show that early afterdepolarization occurs more readily with increased late sodium current compared to decreased repolarization reserve even though action potential durations are similarly prolonged. Interesting to a focal view of arrhythmia described later on, stimulating Gαq receptors greatly increases the frequency at which early afterdepolarization occurs in muscles with decreased repolarization reserve. The molecular basis for this curious effect has not been conclusively established. Early after‐ depolarization occurs most often at low rates of muscle stimulation and materializes much less frequently as the stimulation rate increases toward normal. Thus arrhythmia that arises in settings of bradycardia or in conditions where heart rate is highly variable is often ascri‐ bed to early afterdepolarization. In addition, early afterdepolarization is a likely source for premature atrial (*or ventricular*) contraction and more complex arrhythmia when genetic mu‐ tation or pharmacological intervention prolongs the myocardial QT interval.

Delayed afterdepolarization is the second type of triggered activity. By contrast to early af‐ terdepolarization, muscle or myocytes with normal action potentials that have returned to their Phase IV resting potential generate this type of abnormal impulse. Delayed afterdepo‐ larization usually arises following high frequency burst stimulation of heart or myocytes or when heart calcium stores are greatly increased. Depending on the precise experimental condition, afterdepolarization can occur as a solitary event, as a few afterdepolarizations or as ectopy that lasts for seconds or longer. This latter type of event has been termed 'sus‐ tained triggered activity' [33]. Hypotheses for afterdepolarization must explain isolated events, sustained activity, and the transition between the two. That is, how can a single iso‐ lated ectopic event lead to sustained tachycardic or fibrillary activity?

Schmitt and Erlanger initially explained premature contraction of intact muscle using the impulse reentry hypothesis [35]. In their view, electrical impulses might recirculate through junctions in the Purkinje system or around a region of the heart if both somehow came to possess unidirectional impulse block and altered conduction properties. They envisioned a scenario wherein recirculation could occur once or in a sustained manner depending on the electrical characteristics of the recirculating loop. The observation of afterdepolarization in isolated myocytes indicated that mechanisms besides the gross physiological ones of reentry might also initiate triggered activity. January and others [36] proposed voltage-dependent sodium or calcium channel window currents as potential mediators of early afterdepolariza‐ tion. In their view, the biophysical properties of these voltage-dependent ion channels favor channel reopening during their prolonged exposure to the membrane potentials of the ac‐ tion potential plateau phase. For a wide range of reasons reviewed by Salama and others [37,38], neither of these purely electrical explanations adequately explain the production or the properties of early afterdepolarizations. Window currents also appear to be a less likely explanation for delayed afterdepolarizations which occur from resting potentials. Pogwizd among others [39] hypothesized that decreased activity of the inwardly rectifying potassium channel could sensitize heart muscle to depolarizing influences during diastole. This en‐ hanced sensitivity would favor myocyte delayed afterdepolarization during Phase IV. All of these explanations, however, view afterdepolarization as essentially an electrical phenomen‐ on. That is, they hold that the voltage-dependent ion channels which produce normal elec‐ trical activity are the sole cause for the ectopic electrical instability of afterdepolarization. An alternate view of afterdepolarization began to evolve from data first reported in 2000 [40,41] which proposed that abnormalities in the calcium homeostasis responsible for muscle con‐ traction might cause afterdepolarization.

um release channel, locks it into an open state, and permits the leakage of SR calcium. Us‐ ing ryanodine binding as a molecular probe, they identified and purified the ryanodine receptor calcium release channel and demonstrated its central role in calcium-induced cal‐ cium release. The development of reporter molecules that measure intracellular free calci‐ um, molecules such as aequorin by Blinks [52] and fura-2 by Grynkiewicz and Tsien [53], allowed the interrogation of the intracellular calcium dynamics of cardiac calcium-in‐

Voltage-Independent Calcium Channels, Molecular Sources of Supraventricular Arrhythmia

http://dx.doi.org/10.5772/53649

87

This myocyte calcium-handling system also offers a cell-based mechanism for triggered ac‐ tivity. Marks [40] and then others [41,54,55], proposed that slow leakage of SR calcium through dysfunctional ryanodine receptors might incite afterdepolarization especially de‐ layed afterdepolarization. This 'calcium leak' hypothesis for triggered arrhythmia (Figure 2) takes advantage of the localization of the ventricular SR ryanodine receptor calcium release channel in SR terminal cisternae near the myocyte T-tubule. It posits that SR calcium leak stimulates calcium efflux on the electrogenic sodium-calcium exchanger which would depo‐ larize myocytes during diastole. Thus conditions that (a) increase the content of myocyte cal‐ cium stores, (b) create a steady-state leak of SR calcium or (c) create a preferential leak of calcium during diastole would raise myocyte resting membrane potential to more positive values and reach threshold. Delayed aftedepolarization would result. To some degree this general model may also hold in atrial myocytes that lack well developed T-tubules. Here junctional ryanodine receptors appose the atrial myocyte plasma membranes [54]. Increases in ryanodine receptor calcium leak have been reported in experimentally and pathologically challenged atrial myocytes, indicating that calcium leak might be a generally applicable cause for delayed afterdepolarization. How the disruption of calcium homeostasis generates

'Hyperphosphorylation' of the ryanodine receptor is proposed to incite its leakiness. Using experimental systems as diverse as lipid bilayers and failing hearts the inventive work of Marks [40] and others supported protein kinase A as the agent that hyperphosphorylates the ryanodine receptor and causes leakiness. Work from the laboratory of Bers [41] and others [54,55] highlighted isoforms of calmodulin-dependent protein kinase II (CaMKII) as a sec‐ ond potential initiator of ryanodine receptor hyperphosphorylation/leakiness. The ryano‐ dine receptor is a large protein critical to the normal function of heart muscle. Thus it is not unexpected that many cell factors regulate its properties including its leakiness; redox stress and the interactions between the FKBP12.6 protein and the ryanodine receptor are two such

**5. Germane questions about 'calcium leak' & afterdepolarization**

Several questions arise about the logical & widely accepted calcium-leak hypothesis for trig‐

Does the accepted axis of [SR calcium leakage→electrogenic calcium efflux] describe the en‐ tire mechanism for afterdepolarization or does afterdepolarization result from more compli‐

early afterdepolarization remains under active investigation.

duced calcium release.

factors [40,56].

gered arrhythmia.

The mechanism which couples myocyte excitation and contraction remained unresolved in‐ to the 1970s [42]. The experiments of Fabiato established that the passage of small amounts of calcium across the myocyte plasma membrane initiated the rapid release of a much larger myocyte calcium store sequestered within the lumen of the sarcoplasmic reticulum (SR) [43]. This calcium release causes the rapid elevation of cytosolic free calcium which induces myo‐ filaments to shorten. The subsequent accumulation of this free cytosolic calcium back into the SR lumen promotes muscle relaxation. This process of calcium-induced calcium release is the mechanism through which myocyte electrical depolarization promotes contraction. Particularly important details of this process were provided by the molecular and electro‐ physiological studies of the voltage-dependent slow calcium channel by Fleckenstein and others [44], the SR ryanodine receptor calcium release channel by Fleischer and others [45], and the SR calcium ATPase by MacLennan, Katz, Tada, and others [46-49].

Beta-adrenergic receptor stimulation provokes the phosphorylation of several myocyte pro‐ teins critical for excitation-contraction coupling including SR phospholamban. Phosphoryla‐ tion of phospholamban dissociates it from the SR calcium ATPase which activates this transporter and enhances the sequestration of cytosolic calcium into the SR lumen [48]. As a result, SR calcium stores increase which contributes both to the positive inotropic effect of beta-adrenergic stimulation and to the production of delayed afterdepolarizations. Myocyte calcium stores likewise increase in response to increased cytosolic sodium, for example fol‐ lowing exposure to the Na/K-ATPase inhibitor ouabain. Excess sodium exits myocytes via the plasma membrane sodium-calcium exchange transporter leading to myocyte calcium loading. As first quantitated by Pitts and by Reeves [50,51], this transporter facilitates the electrogenic exchange of three sodium ions for one calcium ion.

Fleischer [45] and others defined the mechanism through which calcium egresses from the SR. They demonstrated that the alkaloid ryanodine binds with high affinity to an SR calci‐ um release channel, locks it into an open state, and permits the leakage of SR calcium. Us‐ ing ryanodine binding as a molecular probe, they identified and purified the ryanodine receptor calcium release channel and demonstrated its central role in calcium-induced cal‐ cium release. The development of reporter molecules that measure intracellular free calci‐ um, molecules such as aequorin by Blinks [52] and fura-2 by Grynkiewicz and Tsien [53], allowed the interrogation of the intracellular calcium dynamics of cardiac calcium-in‐ duced calcium release.

might also initiate triggered activity. January and others [36] proposed voltage-dependent sodium or calcium channel window currents as potential mediators of early afterdepolariza‐ tion. In their view, the biophysical properties of these voltage-dependent ion channels favor channel reopening during their prolonged exposure to the membrane potentials of the ac‐ tion potential plateau phase. For a wide range of reasons reviewed by Salama and others [37,38], neither of these purely electrical explanations adequately explain the production or the properties of early afterdepolarizations. Window currents also appear to be a less likely explanation for delayed afterdepolarizations which occur from resting potentials. Pogwizd among others [39] hypothesized that decreased activity of the inwardly rectifying potassium channel could sensitize heart muscle to depolarizing influences during diastole. This en‐ hanced sensitivity would favor myocyte delayed afterdepolarization during Phase IV. All of these explanations, however, view afterdepolarization as essentially an electrical phenomen‐ on. That is, they hold that the voltage-dependent ion channels which produce normal elec‐ trical activity are the sole cause for the ectopic electrical instability of afterdepolarization. An alternate view of afterdepolarization began to evolve from data first reported in 2000 [40,41] which proposed that abnormalities in the calcium homeostasis responsible for muscle con‐

The mechanism which couples myocyte excitation and contraction remained unresolved in‐ to the 1970s [42]. The experiments of Fabiato established that the passage of small amounts of calcium across the myocyte plasma membrane initiated the rapid release of a much larger myocyte calcium store sequestered within the lumen of the sarcoplasmic reticulum (SR) [43]. This calcium release causes the rapid elevation of cytosolic free calcium which induces myo‐ filaments to shorten. The subsequent accumulation of this free cytosolic calcium back into the SR lumen promotes muscle relaxation. This process of calcium-induced calcium release is the mechanism through which myocyte electrical depolarization promotes contraction. Particularly important details of this process were provided by the molecular and electro‐ physiological studies of the voltage-dependent slow calcium channel by Fleckenstein and others [44], the SR ryanodine receptor calcium release channel by Fleischer and others [45],

Beta-adrenergic receptor stimulation provokes the phosphorylation of several myocyte pro‐ teins critical for excitation-contraction coupling including SR phospholamban. Phosphoryla‐ tion of phospholamban dissociates it from the SR calcium ATPase which activates this transporter and enhances the sequestration of cytosolic calcium into the SR lumen [48]. As a result, SR calcium stores increase which contributes both to the positive inotropic effect of beta-adrenergic stimulation and to the production of delayed afterdepolarizations. Myocyte calcium stores likewise increase in response to increased cytosolic sodium, for example fol‐ lowing exposure to the Na/K-ATPase inhibitor ouabain. Excess sodium exits myocytes via the plasma membrane sodium-calcium exchange transporter leading to myocyte calcium loading. As first quantitated by Pitts and by Reeves [50,51], this transporter facilitates the

Fleischer [45] and others defined the mechanism through which calcium egresses from the SR. They demonstrated that the alkaloid ryanodine binds with high affinity to an SR calci‐

and the SR calcium ATPase by MacLennan, Katz, Tada, and others [46-49].

electrogenic exchange of three sodium ions for one calcium ion.

traction might cause afterdepolarization.

86 Atrial Fibrillation - Mechanisms and Treatment

This myocyte calcium-handling system also offers a cell-based mechanism for triggered ac‐ tivity. Marks [40] and then others [41,54,55], proposed that slow leakage of SR calcium through dysfunctional ryanodine receptors might incite afterdepolarization especially de‐ layed afterdepolarization. This 'calcium leak' hypothesis for triggered arrhythmia (Figure 2) takes advantage of the localization of the ventricular SR ryanodine receptor calcium release channel in SR terminal cisternae near the myocyte T-tubule. It posits that SR calcium leak stimulates calcium efflux on the electrogenic sodium-calcium exchanger which would depo‐ larize myocytes during diastole. Thus conditions that (a) increase the content of myocyte cal‐ cium stores, (b) create a steady-state leak of SR calcium or (c) create a preferential leak of calcium during diastole would raise myocyte resting membrane potential to more positive values and reach threshold. Delayed aftedepolarization would result. To some degree this general model may also hold in atrial myocytes that lack well developed T-tubules. Here junctional ryanodine receptors appose the atrial myocyte plasma membranes [54]. Increases in ryanodine receptor calcium leak have been reported in experimentally and pathologically challenged atrial myocytes, indicating that calcium leak might be a generally applicable cause for delayed afterdepolarization. How the disruption of calcium homeostasis generates early afterdepolarization remains under active investigation.

'Hyperphosphorylation' of the ryanodine receptor is proposed to incite its leakiness. Using experimental systems as diverse as lipid bilayers and failing hearts the inventive work of Marks [40] and others supported protein kinase A as the agent that hyperphosphorylates the ryanodine receptor and causes leakiness. Work from the laboratory of Bers [41] and others [54,55] highlighted isoforms of calmodulin-dependent protein kinase II (CaMKII) as a sec‐ ond potential initiator of ryanodine receptor hyperphosphorylation/leakiness. The ryano‐ dine receptor is a large protein critical to the normal function of heart muscle. Thus it is not unexpected that many cell factors regulate its properties including its leakiness; redox stress and the interactions between the FKBP12.6 protein and the ryanodine receptor are two such factors [40,56].
