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

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

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‐

events specific to alpha-adrenergic receptor stimulation/Gαq signaling might also exacer‐

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**•** Can the stimulation of Gαq-coupled signaling by means other than the alpha-1 receptor enhance afterdepolarization in isolated pulmonary veins or in hearts with reduced repo‐ larization reserve? There is evidence indicating this is the case [61]. Pharmacological and molecular dissection of the interaction between voltage-independent Gαq-coupled signal‐ ing and early- or delayed-afterdepolarization might reveal new mechanisms for arrhyth‐

**•** Does alpha-adrenergic stimulation of Purkinje fibers, isolated pulmonary veins, and nor‐ mal heart muscle with reduced repolarization reserve 'hyperphosphorylate' ryanodine re‐ ceptors compared to normal preparations. If 'hyperphosphorylation' were not to occur,

**•** In the particular case of the pulmonary veins, does calcium loading by approaches other than beta-adrenergic stimulation, approaches like slow calcium channel activation, pro‐

**•** Does 'arrhythmogenic' calcium activate afterdepolarization solely as a charge carrier or as a signaling intermediate that accelerates ryanodine receptor calcium leak? Anderson and co-authors reported in 1998 [62] that CaMKII inhibitors prevent afterdepolarization in in‐ tact and isolated cardiac preparations, a result since widely validated [63]. Whether CaM‐ KII acts by hyperphosphorylating the ryanodine receptor or whether it has multiple

**•** What source of calcium activates CaMKII to provoke triggered activity? Is this source cal‐ cium leaked from the SR or might alternate mean exist to activate arrhythmogenic calmo‐

Triggered afterdepolarization often begins as an isolated event but evolves into more robust and continuous ectopy, so-called sustained triggered activity. This transition depends on the duration of high-frequency burst pacing, the dose of pharmacological activators of the late sodium current, or the apparent timing of R-on-T phenomena. How does the transition from afterdepolarization to complex arrhythmia like tachycardia or fibrillation actually occur? It is now generally accepted that these transitions arise from abnormalities in impulse conduc‐ tion. In this view, ectopic afterdepolarization triggers reentry in arrhythmogenic 'substrate,' heart muscle that conducts impulses heterogeneously. This facile explanation, however, may not address all potential causes for the transition from isolated to complex ectopy. Might afterdepolarization and sustained activity be manifestations of a common cell ar‐

Do both 'isolated' and 'sustained' triggered activities require CaMKII signaling? That is, could myocytes or Purkinje cells express an arrhythmogenic pathway in which CaMKII and afterdepolarization lie upstream of a second calcium-linked mechanism whose stimulation

then additional molecular mechanisms contribute to afterdepolarization.

bate afterdepolarization in hearts with reduced repolarization reserve.

mia.

voke spontaneous ectopic activity?

dulin and CaMKII?

rhythmogenic signaling pathway?

elicits CaMKII-independent 'sustained' ectopic activity?

arrhythmogenic targets remains open to investigation.

**Figure 2. Model for 'Calcium Leak' Afterdepolarization***. Left:* Ryanodine receptors (RyR) are impermeant to calcium except during the action potential when 'trigger calcium' enters myocytes via the voltage-dependent calcium channel (*SCC*). *Right:* Hyper-phosphorylated ryanodine receptors are leaky to calcium. This depletes SR stores. Leaked calcium leaves myocytes on the sodium calcium exchanger (*NCX*). Electrogenic calcium efflux drives positive charges into the myocyte which acts as a depolarizing influence. At impulse threshold, afterdepolarization would occur.

cated molecular pathways? Numerous observations in the literature support the latter view. For example, Ben-David and Zipes [57] showed that reduced repolarization reserve effec‐ tively prolongs the action potential duration of intact heart but does not produce a high inci‐ dence of arrhythmia. By contrast, alpha-adrenergic agonists provoke fulminant early afterdepolarization and complex arrhythmia in intact hearts with low repolarization reserve. Beta-adrenergic stimulation of these hearts does not provoke arrhythmia. Both Kimura and co -authors and Molina-Viamonte and colleagues [58,59] reported that alpha-adrenergic stimulation provoked delayed afterdepolarization in calcium loaded or ischemic Purkinje fi‐ bers. These authors concluded that a specific alpha 1-adrenergic pathway is involved in in‐ ducing triggered activity in the setting of ischemia and reperfusion. Finally Lo and coauthors [60] among others report that alpha- and beta-adrenergic receptor stimulation provokes afterdepolarization in intact pulmonary veins and that CaMKII inhibitors block this triggered activity. While SR calcium leak might account for these results, one or more events specific to alpha-adrenergic receptor stimulation/Gαq signaling might also exacer‐ bate afterdepolarization in hearts with reduced repolarization reserve.


Triggered afterdepolarization often begins as an isolated event but evolves into more robust and continuous ectopy, so-called sustained triggered activity. This transition depends on the duration of high-frequency burst pacing, the dose of pharmacological activators of the late sodium current, or the apparent timing of R-on-T phenomena. How does the transition from afterdepolarization to complex arrhythmia like tachycardia or fibrillation actually occur? It is now generally accepted that these transitions arise from abnormalities in impulse conduc‐ tion. In this view, ectopic afterdepolarization triggers reentry in arrhythmogenic 'substrate,' heart muscle that conducts impulses heterogeneously. This facile explanation, however, may not address all potential causes for the transition from isolated to complex ectopy. Might afterdepolarization and sustained activity be manifestations of a common cell ar‐ rhythmogenic signaling pathway?

cated molecular pathways? Numerous observations in the literature support the latter view. For example, Ben-David and Zipes [57] showed that reduced repolarization reserve effec‐ tively prolongs the action potential duration of intact heart but does not produce a high inci‐ dence of arrhythmia. By contrast, alpha-adrenergic agonists provoke fulminant early afterdepolarization and complex arrhythmia in intact hearts with low repolarization reserve. Beta-adrenergic stimulation of these hearts does not provoke arrhythmia. Both Kimura and co -authors and Molina-Viamonte and colleagues [58,59] reported that alpha-adrenergic stimulation provoked delayed afterdepolarization in calcium loaded or ischemic Purkinje fi‐ bers. These authors concluded that a specific alpha 1-adrenergic pathway is involved in in‐ ducing triggered activity in the setting of ischemia and reperfusion. Finally Lo and coauthors [60] among others report that alpha- and beta-adrenergic receptor stimulation provokes afterdepolarization in intact pulmonary veins and that CaMKII inhibitors block this triggered activity. While SR calcium leak might account for these results, one or more

myocyte which acts as a depolarizing influence. At impulse threshold, afterdepolarization would occur.

**Figure 2. Model for 'Calcium Leak' Afterdepolarization***. Left:* Ryanodine receptors (RyR) are impermeant to calcium except during the action potential when 'trigger calcium' enters myocytes via the voltage-dependent calcium channel (*SCC*). *Right:* Hyper-phosphorylated ryanodine receptors are leaky to calcium. This depletes SR stores. Leaked calcium leaves myocytes on the sodium calcium exchanger (*NCX*). Electrogenic calcium efflux drives positive charges into the

RyR

Ca

Ca

**SCC**

88 Atrial Fibrillation - Mechanisms and Treatment

**SERCA**

**Intact ryanodine receptor Electrically** *stable* **myocyte**

Ca

Ca

Ca

Ca Ca

Ca

Ca

**NCX**

3Na

**NCX**

Ca 3Na **+** Ca

Ca

Ca

RyR

**SCC**

**SERCA**

**Hyper-phosphorylatedryanodine receptor ( ) Leaky to calcium ( ) Electrically** *unstable* **myocyte**

Ca

Ca

Ca

Ca

Ca

Ca

Do both 'isolated' and 'sustained' triggered activities require CaMKII signaling? That is, could myocytes or Purkinje cells express an arrhythmogenic pathway in which CaMKII and afterdepolarization lie upstream of a second calcium-linked mechanism whose stimulation elicits CaMKII-independent 'sustained' ectopic activity?

TRP channels, the Orai channels, and Stim1. This signaling system normally regulates the Gαq-coupled growth response, stress responses, and other events in all cells including myo‐ cytes. Our data and that of others lead to an initial hypothesis that voltage-independent cal‐ cium signaling assumes an additional, apparently untoward task in cells like myocytes or Purkinje cells that highly express voltage-dependent ion channels. This task is the activation of a calcium-dependent arrhythmogenic signaling pathway. This putative pathway is nor‐ mally silent until appropriate arrhythmogenic stimuli or pharmacological activators rouse it into action. Depending on the intensity of the activation challenge, we believe this complex pathway can co-opt the activity of voltage-dependent ion channels to produce isolated after‐ depolarization, afterdepolarization that leads to sustained activity, and high frequency sus‐ tained ectopic activity. In this view, solitary afterdepolarizations are focal events that result from the activation of one part of a broader calcium-dependent arrhythmogenic pathway. The activation of an interrelated downstream part of this pathway provokes high-frequency focal tachycardia or fibrillation. The two parts of this putative pathway functionally interact which allows the transition between afterdepolarization and complex arrhythmia. This in‐ teraction might transpire in a manner analogous to that described by Shuttleworth [64] for the sequential activation of voltage-independent calcium signaling and calcium entry path‐ ways in non-excitable cells. In heart the putative calcium signaling events that cause afterde‐ polarization would gradually deplete cell voltage-independent calcium stores specific for calcium signaling. This depletion stimulates voltage-independent calcium entry via the Orai channels. We suggest that this type of calcium entry activates sustained ectopic activity.

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Abnormal automaticity occurs when ectopic sites in the atria (*or ventricle*) spontaneously de‐ polarize independently of normal sinus rhythm or without a preceding triggering event. In‐ vestigators like Vassalle [65] have made important contributions to our current understanding of this type of ectopy. Typical abnormal automaticity occurs during hypoxia and ischemia when myocytes partially depolarize from their resting potential of ~-85 to about -65mV. An additional mechanism for abnormal automaticity takes advantage of the fact that the hyperpolarization-activated 'funny currents', which contribute to normal auto‐ maticity, are expressed throughout the heart [66]. The activation of atrial or ventricular fun‐ ny currents might induce spontaneous depolarization akin to the sinoatrial pacemaker but the properties of these ectopic channels indicate that they are inactive in normal myocytes. How ectopically expressed funny channels might spring to life to provoke focal abnormal

Several reports suggest the existence of alternate, atypical forms of abnormal automaticity and that atypical automaticity may be an unrecognized contributor to arrhythmogenesis. For example, in 1999 Nuss and co-workers [67] reported that myocytes isolated from failing hearts produced sporadic, spontaneous depolarizations while normal myocytes did not. These ectopic depolarizations occurred from normal resting potentials, did not require a preceding external stimulation, and occurred independently of any significant change in in‐

**6. Mechanism 3: Typical abnormal automaticity**

automaticity is unresolved.

**Figure 3. Four Families of Voltage-Independent Calcium Channels.***Left:* IP3Rs allow calcium release from intracellu‐ lar ER/SR stores. This generates intracellular signals. ER store depletion activates calcium entry via the Orail a/o Orai1/ TRPC1 store-operated calcium channel (*Left box*). *Center:* The transient receptor potential channels permit calcium en‐ try into cells in response to a wide range of influences pertinent to atrial fibrillation (*Middle box*). These calcium signals mediate the phenotypic response of atria to stretch or to autonomic signaling. *Right:* Orail and Orai3 create an arach‐ idonate-sensitive calcium channel. This channel permits calcium entry in response to stress signals that activate eicosa‐ noid metabolism (*Right box*).

The well-documented role of calcium in arrhythmogenesis and the central role of SR calci‐ um in heart muscle contraction focused the 'calcium leak' hypothesis on the SR ryanodine receptor as the source of arrhythmogenic calcium. At the time of its formulation only the ryanodine receptor, the voltage-dependent slow calcium channel, and the sodium-calcium exchanger were accepted to greatly affect cytosolic calcium in atrial or ventricular myocytes and Purkinje cells. Now extensive work in non-excitable cells has established the voltageindependent inositol-tris-phosphate receptors (IP3R), the transient receptor potential protein (TRP) channels and the Orai channels are the predominant means to generate cell calcium signals (Figure 3).

*Stating our proposition succinctly, do after depolarization and complex arrhythmia arise from cell processes other than those which produce excitation and the ECG (voltage-dependent ion channels) or myocyte contraction (calcium-induced SR calcium release)? Might myocardial non-electrical, volt‐ age-independent processes provoke myocardial electrical instability including after depolarization?*

Reports in the literature and our data suggest they do. Myocytes and Purkinje cells express the cellular calcium transporters, kinases, lipases and other proteins that initiate and regu‐ late voltage-independent calcium entry and calcium signaling. These include the IP3Rs, the TRP channels, the Orai channels, and Stim1. This signaling system normally regulates the Gαq-coupled growth response, stress responses, and other events in all cells including myo‐ cytes. Our data and that of others lead to an initial hypothesis that voltage-independent cal‐ cium signaling assumes an additional, apparently untoward task in cells like myocytes or Purkinje cells that highly express voltage-dependent ion channels. This task is the activation of a calcium-dependent arrhythmogenic signaling pathway. This putative pathway is nor‐ mally silent until appropriate arrhythmogenic stimuli or pharmacological activators rouse it into action. Depending on the intensity of the activation challenge, we believe this complex pathway can co-opt the activity of voltage-dependent ion channels to produce isolated after‐ depolarization, afterdepolarization that leads to sustained activity, and high frequency sus‐ tained ectopic activity. In this view, solitary afterdepolarizations are focal events that result from the activation of one part of a broader calcium-dependent arrhythmogenic pathway. The activation of an interrelated downstream part of this pathway provokes high-frequency focal tachycardia or fibrillation. The two parts of this putative pathway functionally interact which allows the transition between afterdepolarization and complex arrhythmia. This in‐ teraction might transpire in a manner analogous to that described by Shuttleworth [64] for the sequential activation of voltage-independent calcium signaling and calcium entry path‐ ways in non-excitable cells. In heart the putative calcium signaling events that cause afterde‐ polarization would gradually deplete cell voltage-independent calcium stores specific for calcium signaling. This depletion stimulates voltage-independent calcium entry via the Orai channels. We suggest that this type of calcium entry activates sustained ectopic activity.
