**9. Is voltage-independent calcium signaling a focal source of arrhythmia?**

Experimental evidence acquired in intact animals, in intact heart muscle, and intact pulmo‐ nary veins coupled with clinical studies of human arrhythmia strongly suggest that Gαqcoupled receptor stimulation and by inference voltage-independent calcium signaling can initiate afterdepolarization and more complex arrhythmia. However, no attempt was made in these intact preparations to positively connect the calcium signaling linked to IP3Rs, the TRP channels or the Orai channels to atrial electrical instability.

Bootman and Blatter [84,91] acquired such evidence in isolated atrial myocytes. They dem‐ onstrated that Gαq agonists like endothelin-1 and pharmacological activators of the IP3Rs provoke ectopic calcium sparks, calcium waves, spontaneous calcium transients, and calci‐ um alternans in atrial myocytes. Both groups concluded that exuberant calcium release from IP3Rs sensitizes the junctional ryanodine receptors of atrial myocytes, increasing their sus‐ ceptibility to spontaneous calcium release events. Importantly, low concentrations of 2APB that block both the IP3Rs and the TRP channels suppress abnormal atrial myocyte calcium release. Blatter then showed [92] that the genetic ablation of the atrial myocyte type 2 IP3R suppresses 'arrhythmogenic' calcium release in atrial myocytes treated with endothelin-1. Together these data support and extend earlier intact animal studies and provide striking evidence that voltage-independent calcium homeostasis contributes to atrial arrhythmogen‐ ic calcium signaling.

Purkinje cells. Here they associate with ryanodine receptors within specific regions of the cytoplasm just below the Purkinje plasma membrane. Boyden, ter Keurs and co-authors speculate that this striking arrangement plays a role in the arrhythmogenic potential of the conduction system. Establishing this critically important conclusion is a clear priority in ar‐ rhythmia research. Little is known about Purkinje cell expression of the TRP channels, the Orai channels or Stims. One could speculate that Stim1, Orai1 and TRPC1 might be highly expressed in the conduction system as they are functionally related to the IP3Rs. One ques‐ tion of potential importance is whether the marked increase in IP3R expression reported in failing heart occurs in the Purkinje system, in myocytes or in both. Furthermore, it would be useful to determine whether the expression of Orai1, Stim1, and TRPC1 respond similarly to 'failure' as do the IP3Rs. If the expression of these three IP3R partners were to increase, then the activity or hyperactivity of voltage-independent calcium signaling may contribute to the increased arrhythmogenicity seen in heart failure, as Boyden and ter Keurs speculate [88].

Ju and co-authors [89] and Demion and co-authors [90] reported that the sinoatrial node expresses the TRP channels which play a role in normal automaticity. A more detailed analysis of the expression of other voltage-independent calcium signaling proteins and how they contribute to normal automaticity is clearly required. To our knowledge noth‐ ing is known of the expression or activity of voltage-independent calcium signaling pro‐ teins in the muscular sleeves of the pulmonary or other supraventricular vessels. Since alpha-adrenergic agonists induce afterdepolarization and automatic activity in these ana‐ tomical structures, characterizing 'muscular sleeve' TRP channel, Orai channel, Stim, and IP3R expression should aid in establishing whether these channels contribute to paroxys‐

**9. Is voltage-independent calcium signaling a focal source of arrhythmia?**

Experimental evidence acquired in intact animals, in intact heart muscle, and intact pulmo‐ nary veins coupled with clinical studies of human arrhythmia strongly suggest that Gαqcoupled receptor stimulation and by inference voltage-independent calcium signaling can initiate afterdepolarization and more complex arrhythmia. However, no attempt was made in these intact preparations to positively connect the calcium signaling linked to IP3Rs, the

Bootman and Blatter [84,91] acquired such evidence in isolated atrial myocytes. They dem‐ onstrated that Gαq agonists like endothelin-1 and pharmacological activators of the IP3Rs provoke ectopic calcium sparks, calcium waves, spontaneous calcium transients, and calci‐ um alternans in atrial myocytes. Both groups concluded that exuberant calcium release from IP3Rs sensitizes the junctional ryanodine receptors of atrial myocytes, increasing their sus‐ ceptibility to spontaneous calcium release events. Importantly, low concentrations of 2APB that block both the IP3Rs and the TRP channels suppress abnormal atrial myocyte calcium release. Blatter then showed [92] that the genetic ablation of the atrial myocyte type 2 IP3R suppresses 'arrhythmogenic' calcium release in atrial myocytes treated with endothelin-1.

TRP channels or the Orai channels to atrial electrical instability.

mal atrial fibrillation.

98 Atrial Fibrillation - Mechanisms and Treatment

The depletion of inositol-1,4,5-trisphosphate sensitive calcium stores which likely occurs with high levels of Gαq stimulation provokes SOCC calcium entry [64,78,81,82]. Thus while disturbed inositol-1,4,5-trisphosphate-linked calcium signaling is arrhythmogenic, it re‐ mains open to question whether (a) calcium release through IP3Rs, (b) the attendant in‐ crease in SOCC calcium entry or (c) both provoke ectopy. Furthermore whether these ectopic calcium release events produce myocyte depolarization in a 1:1 manner remains to be established as well as the mechanism through which ectopic depolarization might occur. It is also important to define whether the cause for abnormal depolarization in these myo‐ cytes is solely or mainly calcium efflux on the sodium-calcium exchanger or if other calcium signaling events are involved.

Hirose and co-authors [93] used transgenesis to obtain molecular and pharmacological evi‐ dence that dysregulated Gαq-coupled calcium signaling profoundly disrupts atrial and ven‐ tricular electrical stability. They employed a mouse model developed by Mende [94] which transiently overexpresses constitutively active Gαq in a heart-specific manner. The atria of these genetically modified mice are grossly enlarged and exhibit paroxysmal or persistent fibrillation. To establish that deranged diacylglycerol metabolism caused these atrial abnor‐ malities, Hirose created a second mouse which overexpresses both Gαq and diacylglycerol kinase ζ. Such a double transgenic would accelerate diacylglycerol phosphorylation to phos‐ phatidic acid, reduce heart content of diacylglycerol, and thus TRPC3 signaling. Mice har‐ boring both transgenes had essentially normal atrial anatomy and electrical activity. The current reentry hypothesis for atrial fibrillation would propose that the electrical instability observed in the atria of Gαq overexpressors results from atrial enlargement and from the high levels of fibrosis observed in these muscles. In this electrocentric view, transgenically increasing diacylglycerol kinase activity would suppress atrial fibrillation by restoring nor‐ mal atrial size and by reducing arrhythmogenic atrial scarring/abnormal conduction. Curi‐ ously, reentry also proposes electrical abnormalities like fibrillation should not occur in muscles as small as mouse atria (*or ventricle*) [20,22,24]. Vaidya and authors [95] first report‐ ed a similar egregious violation of Garrey's 'critical mass' tenet for reentry when they re‐ ported the occurrence of faradic fibrillation in mouse heart. They postulated unusual forms of wavebreak to account for this unexpected result.

Hirose and co-authors addressed this possible interpretation of their data in a follow-on pa‐ per [96]. Here they investigated how Gαq overexpression affected ventricular electrical sta‐ bility and heart failure. They observed that mice which overexpress constitutively active Gαq exhibit heart failure and sustained or paroxysmal ventricular tachycardia and fibrilla‐ tion. Some of the ventricular arrhythmia recorded in these transgenic mice may result from the irregularly irregular electrical activity produced by fibrillating atria but much of this ec‐ topy appeared to originate in the ventricles themselves. Importantly, they reported that the acute administration of SKF-96365, a TRP and Orai channel inhibitor [97], reverses ventricu‐ lar fibrillation and restores sinus rhythm in Gαq transgenic mice. This result could only oc‐ cur if SKF-96365 also effectively suppressed atrial fibrillation in these animals. It is vital to remember that the atria of these transgenic mice treated acutely with SKF-96365 remained grossly enlarged and fibrotic. This single result, obtained in a model which mimics the high autonomic drive associated with atrial fibrillation, dissociates fibrillation from atrial enlarge‐ ment and fibrosis.

sustaining and provoked by atrial enlargement and fibrotic substrate, they should not have reversed abruptly or at all. That they did suggests that cell events may indeed drive this ar‐

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101

These data in humans, intact animals, preparations of pulmonary vascular tissue, and in iso‐ lated myocytes pinpoint voltage-independent calcium homeostasis as an underappreciated source of arrhythmia (Figure 6). That is, these types of signaling events when regulated and occurring at normal levels allow hearts to increase mass in response to hypertrophic stimuli. By contrast, the dysregulation or hyperactivity of one or more aspects of voltage-independ‐ ent calcium entry or downstream signaling appears to elicit spontaneous sporadic or high frequency ectopic depolarizations in intact atria and ventricle. Consequently some arrhyth‐ mia might be purely a cell's response to extra- or intra-cellular conditions that disrupt volt‐ age-independent calcium homeostasis. Note that in contrast to 'calcium leak' models which often require burst pacing to induce atrial (*or ventricular*) arrhythmia [40,54,55], the disrup‐ tion of voltage-independent calcium homeostasis results in intact heart muscle spontaneous‐

While provocative these evidences for a focal mechanism for arrhythmia leave unanswered

**•** Which part or parts of voltage-independent calcium homeostasis underlie this arrhythmic activity, (a) calcium release through the IP3R, (b) calcium entry via one of more of the TRP channels, (c) calcium entry via the Orai channels and/or (d) calcium signaling down‐

**•** Can this novel mechanism for arrhythmia account for the gamut of ectopic activities from

**•** How might pathological stimuli or high autonomic activity favor the activation of this ar‐

Work from our laboratory has begun to address these questions using the following rationale. Lewis [98,99], Putney [78], Shuttleworth [64] and others identify voltage-independent calci‐ um homeostasis as a dynamic process that depends on the inter-relationship between multi‐ ple families of calcium channels and the filling state of intracellular calcium stores. In this model Gαq agonists provoke calcium entry via TRPC3 as well as the release of calcium from internal stores regulated by IP3Rs. These calcium entry and release events sum to generate intracellular signals which are then terminated by re-accumulation of calcium into the endo‐ plasmic reticulum lumen. The net flux of calcium out of the reticular lumen is a sum of all inputs experienced by a cell under any particular physiological or pathophysiological condi‐ tions. As one or more of these agonist signals increases in intensity, the local or the net calci‐ um content of the reticular calcium stores begins to decrease. As stores deplete, the [Stim1- Orai1/TRPC1] channel complex activates to refill them, permitting continued calcium signaling. Excessive or continual calcium store depletion initiates a strong SOCC calcium en‐ try response. Earlier studies suggest a potent arrhythmic effect associates with excessive or

**•** What is the final molecular initiator of this putative focal mechanism for arrhythmia?

sporadic depolarization to paroxysmal or sustained tachycardia to fibrillation?

rhythmic activity.

at least four questions.

stream of these channels?

rhythmogenic mechanism?

ly producing profound complex arrhythmia.

Hirose's data argue that a focal, non-reentrant mechanism can produce atrial and ventricu‐ lar fibrillation. Specifically, the genetic activation of Gαq-coupled signaling promotes car‐ diac hypertrophy which would enlarge the atria in Gαq transgenic mice. Atrial fibrosis may result from enhanced Gαq signaling or from the activation of specific gene programs. This transgenic intervention enhances heart diacylglycerol content and consequently the activity of TRPC3/6. Exuberant Gαq stimulation, voltage-independent calcium entry and signaling might deplete or disrupt voltage-independent calcium stores initiating compensatory SOCC calcium entry. The acute administration of SKF-96365 would block calcium entry via TRPC3/6 and/or the Orai1/3 channels. Thus calcium entry via voltage-independent calcium channels or arrhythmogenic signaling events downstream of these channels may cause atrial and ventricular fibrillation in this model.

excitation-contraction coupling in normal cells while voltage-independent calcium signaling controls growth and apoptosis. *Right*: **Arrhythmogenic-** In stressed cells voltage-independent calcium signaling subsumes a novel, untoward function. It co-opts voltage-dependent ion channels to act independently of external electrical impulses and produce high frequency ectopic depolarizations. These arrhythmogenic foci of myocytes electrically capture the heart, subvert organized 'sinus rhythm' & cause arrhythmia. **Figure 6. General Model for a Voltage-Independent Mechanism for Arrhythmia.***Left:***Normal:** - Voltage-depend‐ ent ion channels regulate excitation-contraction coupling in normal cells while voltage-independent calcium signaling controls growth and apoptosis. *Right:* **Arrhythmogenic**: In stressed cells, voltage-independent calcium signaling sub‐ serves a novel, untoward function. It co-opts voltage-dependent ion channels to act independently of external electri‐ cal impulses and produce high frequency ectopic depolarizations. These arrhythmogenic foci of myocytes electrically capture the heart, subvert organized sinus rhythm & cause arrhythmia.

**Figure 6**: **General Model for a Voltage-Independent Mechanism for Arrhythmia**. *Left*: **Normal** - Voltage-dependent ion channels regulate

The reentry hypothesis would propose that rhythm disturbances in Gαq overexpressing mice occur because hypertrophy and fibrosis provide an 'arrhythmogenic substrate' that in‐ homogeneously conducts electrical impulses. In a reentrant view fibrosis, hypertrophy, and arrhythmia cannot be completely dissociated. By contrast, a focal view proposes that ar‐ rhythmia arises from cell signaling events that may be functionally distinct from those that produce fibrosis or hypertrophy; these three events may be dissociable. Hirose's SKF-96365 data support a focal view. If the atrial and ventricular fibrillation in these mice were selfsustaining and provoked by atrial enlargement and fibrotic substrate, they should not have reversed abruptly or at all. That they did suggests that cell events may indeed drive this ar‐ rhythmic activity.

cur if SKF-96365 also effectively suppressed atrial fibrillation in these animals. It is vital to remember that the atria of these transgenic mice treated acutely with SKF-96365 remained grossly enlarged and fibrotic. This single result, obtained in a model which mimics the high autonomic drive associated with atrial fibrillation, dissociates fibrillation from atrial enlarge‐

Hirose's data argue that a focal, non-reentrant mechanism can produce atrial and ventricu‐ lar fibrillation. Specifically, the genetic activation of Gαq-coupled signaling promotes car‐ diac hypertrophy which would enlarge the atria in Gαq transgenic mice. Atrial fibrosis may result from enhanced Gαq signaling or from the activation of specific gene programs. This transgenic intervention enhances heart diacylglycerol content and consequently the activity of TRPC3/6. Exuberant Gαq stimulation, voltage-independent calcium entry and signaling might deplete or disrupt voltage-independent calcium stores initiating compensatory SOCC calcium entry. The acute administration of SKF-96365 would block calcium entry via TRPC3/6 and/or the Orai1/3 channels. Thus calcium entry via voltage-independent calcium channels or arrhythmogenic signaling events downstream of these channels may cause atrial

> Sinus excitation

**Voltage-dependent Na/Ca/K ion channels**

**Disrupted voltage-independent Ca++ homeostasis**

**Arrhythmogenic inputs**

**Figure 6**: **General Model for a Voltage-Independent Mechanism for Arrhythmia**. *Left*: **Normal** - Voltage-dependent ion channels regulate excitation-contraction coupling in normal cells while voltage-independent calcium signaling controls growth and apoptosis. *Right*: **Arrhythmogenic-** In stressed cells voltage-independent calcium signaling subsumes a novel, untoward function. It co-opts voltage-dependent ion channels to act independently of external electrical impulses and produce high frequency ectopic depolarizations. These arrhythmogenic foci of

**Figure 6. General Model for a Voltage-Independent Mechanism for Arrhythmia.***Left:***Normal:** - Voltage-depend‐ ent ion channels regulate excitation-contraction coupling in normal cells while voltage-independent calcium signaling controls growth and apoptosis. *Right:* **Arrhythmogenic**: In stressed cells, voltage-independent calcium signaling sub‐ serves a novel, untoward function. It co-opts voltage-dependent ion channels to act independently of external electri‐ cal impulses and produce high frequency ectopic depolarizations. These arrhythmogenic foci of myocytes electrically

The reentry hypothesis would propose that rhythm disturbances in Gαq overexpressing mice occur because hypertrophy and fibrosis provide an 'arrhythmogenic substrate' that in‐ homogeneously conducts electrical impulses. In a reentrant view fibrosis, hypertrophy, and arrhythmia cannot be completely dissociated. By contrast, a focal view proposes that ar‐ rhythmia arises from cell signaling events that may be functionally distinct from those that produce fibrosis or hypertrophy; these three events may be dissociable. Hirose's SKF-96365 data support a focal view. If the atrial and ventricular fibrillation in these mice were self-

**↑** External & Internal inputs

Automatic action potentials

Stress calcium signals

ment and fibrosis.

100 Atrial Fibrillation - Mechanisms and Treatment

Sinus excitation

External & internal inputs

and ventricular fibrillation in this model.

**Voltage-dependent Na/Ca/K ion channels**

**Voltage-independent Ca++ homeostasis**

Non-automatic action potentials

Calcium signals

myocytes electrically capture the heart, subvert organized 'sinus rhythm' & cause arrhythmia.

capture the heart, subvert organized sinus rhythm & cause arrhythmia.

These data in humans, intact animals, preparations of pulmonary vascular tissue, and in iso‐ lated myocytes pinpoint voltage-independent calcium homeostasis as an underappreciated source of arrhythmia (Figure 6). That is, these types of signaling events when regulated and occurring at normal levels allow hearts to increase mass in response to hypertrophic stimuli. By contrast, the dysregulation or hyperactivity of one or more aspects of voltage-independ‐ ent calcium entry or downstream signaling appears to elicit spontaneous sporadic or high frequency ectopic depolarizations in intact atria and ventricle. Consequently some arrhyth‐ mia might be purely a cell's response to extra- or intra-cellular conditions that disrupt volt‐ age-independent calcium homeostasis. Note that in contrast to 'calcium leak' models which often require burst pacing to induce atrial (*or ventricular*) arrhythmia [40,54,55], the disrup‐ tion of voltage-independent calcium homeostasis results in intact heart muscle spontaneous‐ ly producing profound complex arrhythmia.

While provocative these evidences for a focal mechanism for arrhythmia leave unanswered at least four questions.


Work from our laboratory has begun to address these questions using the following rationale.

Lewis [98,99], Putney [78], Shuttleworth [64] and others identify voltage-independent calci‐ um homeostasis as a dynamic process that depends on the inter-relationship between multi‐ ple families of calcium channels and the filling state of intracellular calcium stores. In this model Gαq agonists provoke calcium entry via TRPC3 as well as the release of calcium from internal stores regulated by IP3Rs. These calcium entry and release events sum to generate intracellular signals which are then terminated by re-accumulation of calcium into the endo‐ plasmic reticulum lumen. The net flux of calcium out of the reticular lumen is a sum of all inputs experienced by a cell under any particular physiological or pathophysiological condi‐ tions. As one or more of these agonist signals increases in intensity, the local or the net calci‐ um content of the reticular calcium stores begins to decrease. As stores deplete, the [Stim1- Orai1/TRPC1] channel complex activates to refill them, permitting continued calcium signaling. Excessive or continual calcium store depletion initiates a strong SOCC calcium en‐ try response. Earlier studies suggest a potent arrhythmic effect associates with excessive or

**Figure 7**: M**odel for Orai Arrhythmogenesis**. *Left*: Under normal conditions Orais are tightly regulated and not arrhythmogenic. *Center*: Low level dysregulation (or activation) of Orais by numerous factors (*Lower right box*) will produce a progressive arrhythmic effect. At intermediate levels the calcium signal will provoke aferdepolarization. *Right*: At high levels of Orai opening the calcium signal co-opts myocyte voltage-dependent ion channels to provoke ~20Hz atypical **Figure 7. Model for Orai Arrhythmogenesis.***Left:* Under normal conditions Orais are tightly regulated and not ar‐ rhythmogenic. *Center*: Low level dysregulation (or activation) of Orais by numerous factors (*Lower right box*) will pro‐ duce a progressive arrhythmic effect. At intermediate levels the calcium signal will provoke afterdepolarization. *Right*: At high levels of Orai opening the calcium signal co-opts myocyte voltage-dependent ion channels to provoke ∼20Hz atypical automaticity and fibrillation. 2APB causes automatic tachycardia and fibrillation in this manner.

intact, superfused normal rat left atria and rat left ventricular papillary muscles begin to spontaneously contact when they are challenged with 2APB at concentrations greater than 10μM. This ectopic activity takes several minutes to arise following muscle exposure to 2APB but once initiated it occurs persistently until this borinate is removed from the super‐ fusate. Increasing muscle calcium by several disparate means including slow channel activa‐ tion and ouabain markedly increases the rate of this ectopic activity [104 *see Table I*]. Under well-defined conditions isolated left atria and left ventricular papillaries can produce persis‐ tent ectopic activity at rates of at least 10 to 12 Hz at 37°C (Figure 8). These rates are similar to those reported for arrhythmic drivers of clinical and experimental arrhythmia [107]. Reen‐ trant mechanisms are usually invoked to explain such drivers but cell-based focal means may also exist to provoke persistent high frequency ectopy. The disruption of heart muscle electromechanical stability by 2APB is not self-sustaining as this high frequency ectopy stops immediately after the removal of this molecule from the superfusate. One implication from this reversible destabilization is that a cell-based voltage-independent mechanism for arrhythmia would produce electrical instability only as long as it remains stimulated. Parox‐ ysmal or persistent arrhythmia thus might result if the pathological disturbance of voltageindependent calcium homeostasis were ephemeral or unrelenting in nature regardless of the presence or absence of arrhythmogenic 'substrate.' Hirose's work and our data in fibrillating

**Figure 8. 2APB Activation of 10Hz Atypical Automaticity in Superfused Rat Left Atria.***Left. Upper:* The mechanical function of an unpaced rat right atrium superfused at 37°C. Spontaneous sinoatrial node-driven normal automaticity occurs at —6Hz in this muscle. *Middle:* An unpaced rat left atrial appendage superfused with 300nM Bayk at 37°C. This left atrium and all others do not contract under this condition. *Bottom.* An unpaced rat left atrium superfused with 300nM BayK and 20μM 2APB. This muscle produces spontaneous mechanical activity at 10Hz. *Right.* Summary of groups of unpaced right atria (Δ; n=7) superfused at 23, 30, and 37°C and unpaced left atria superfused with BayK and 20μM 2APB (∎; n=9) at 23, 30, and 37°C. Left atria treated with BayK and 2APB perfused at 37°C spontaneously

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103

2APB induces a unique atypical automaticity in non-automatic heart muscle. Specifically, 2APB provokes high frequency ectopic action potentials and muscle contraction even when added to the superfusate of quiescent left atria or left ventricular papillary muscles [105 *see Figure 3*]. That is, non-automatic heart muscle which normally requires external stimulation to produce action potentials and contract will do both spontaneously, at high-frequency, and in the absence of a triggering depolarizing stimulus if these quiescent muscles are ex‐

hearts discussed in a following section substantiate this speculation.

contract at rates of 543±13 contractions/minute.

automaticity and fibrillation. **2APB** causes automatic tachycardia and fibrillation in this manner.

dysregulated activation of this overall pathway [57-61]. The interpretation of this data fo‐ cused on calcium release through the IP3R and showed that pharmacological blockade or genetic ablation of this protein suppresses ectopic electromechanical activity. However, (a) Gαq stimulation, (b) voltage-independent calcium release from the IP3Rs, and (c) Orailinked SOCC calcium entry are interrelated events [64,98]. Thus exuberant arrhythmogenic Gαq stimulation [96] or IP3R calcium release [91,92] will activate Orai-linked calcium entry (Figure 7). Previous experiments did not fully address whether the first two of these volt‐ age-independent events or the third one, Orai-linked calcium entry, might drive arrhythmic activity. Thus we wished to test whether increased Orai channel opening might be an unrec‐ ognized arrhythmic principle (Figure 7). This requires a means to activate the Orais.

Putney reported [100] that 2-aminoethoxydiphenyl borate (2APB) activates calcium entry in non-excitable cells apparently by a store-operated mechanism. Subsequent work [101-102] conclusively demonstrated that 2APB pharmacologically opens Orai1 with an EC50 of 20μM and Orai3 with an EC50 of 13μM. 2APB also alters the ion conduction properties of these channels to enhance sodium transport. We took advantage of this Orai channel opener to in‐ terrogate in a crude manner whether activating voltage-independent calcium channels might underlie a focal mechanism for arrhythmia.

We found that 2APB provokes a novel type of arrhythmic activity [103-106] which appears to satisfy the demands of the focal source hypothesis of Engelmann and Scherf. In particular,

**Figure 8. 2APB Activation of 10Hz Atypical Automaticity in Superfused Rat Left Atria.***Left. Upper:* The mechanical function of an unpaced rat right atrium superfused at 37°C. Spontaneous sinoatrial node-driven normal automaticity occurs at —6Hz in this muscle. *Middle:* An unpaced rat left atrial appendage superfused with 300nM Bayk at 37°C. This left atrium and all others do not contract under this condition. *Bottom.* An unpaced rat left atrium superfused with 300nM BayK and 20μM 2APB. This muscle produces spontaneous mechanical activity at 10Hz. *Right.* Summary of groups of unpaced right atria (Δ; n=7) superfused at 23, 30, and 37°C and unpaced left atria superfused with BayK and 20μM 2APB (∎; n=9) at 23, 30, and 37°C. Left atria treated with BayK and 2APB perfused at 37°C spontaneously contract at rates of 543±13 contractions/minute.

intact, superfused normal rat left atria and rat left ventricular papillary muscles begin to spontaneously contact when they are challenged with 2APB at concentrations greater than 10μM. This ectopic activity takes several minutes to arise following muscle exposure to 2APB but once initiated it occurs persistently until this borinate is removed from the super‐ fusate. Increasing muscle calcium by several disparate means including slow channel activa‐ tion and ouabain markedly increases the rate of this ectopic activity [104 *see Table I*]. Under well-defined conditions isolated left atria and left ventricular papillaries can produce persis‐ tent ectopic activity at rates of at least 10 to 12 Hz at 37°C (Figure 8). These rates are similar to those reported for arrhythmic drivers of clinical and experimental arrhythmia [107]. Reen‐ trant mechanisms are usually invoked to explain such drivers but cell-based focal means may also exist to provoke persistent high frequency ectopy. The disruption of heart muscle electromechanical stability by 2APB is not self-sustaining as this high frequency ectopy stops immediately after the removal of this molecule from the superfusate. One implication from this reversible destabilization is that a cell-based voltage-independent mechanism for arrhythmia would produce electrical instability only as long as it remains stimulated. Parox‐ ysmal or persistent arrhythmia thus might result if the pathological disturbance of voltageindependent calcium homeostasis were ephemeral or unrelenting in nature regardless of the presence or absence of arrhythmogenic 'substrate.' Hirose's work and our data in fibrillating hearts discussed in a following section substantiate this speculation.

dysregulated activation of this overall pathway [57-61]. The interpretation of this data fo‐ cused on calcium release through the IP3R and showed that pharmacological blockade or genetic ablation of this protein suppresses ectopic electromechanical activity. However, (a) Gαq stimulation, (b) voltage-independent calcium release from the IP3Rs, and (c) Orailinked SOCC calcium entry are interrelated events [64,98]. Thus exuberant arrhythmogenic Gαq stimulation [96] or IP3R calcium release [91,92] will activate Orai-linked calcium entry (Figure 7). Previous experiments did not fully address whether the first two of these volt‐ age-independent events or the third one, Orai-linked calcium entry, might drive arrhythmic activity. Thus we wished to test whether increased Orai channel opening might be an unrec‐

atypical automaticity and fibrillation. 2APB causes automatic tachycardia and fibrillation in this manner.

**Figure 7**: M**odel for Orai Arrhythmogenesis**. *Left*: Under normal conditions Orais are tightly regulated and not arrhythmogenic. *Center*: Low level dysregulation (or activation) of Orais by numerous factors (*Lower right box*) will produce a progressive arrhythmic effect. At intermediate levels the calcium signal will provoke aferdepolarization. *Right*: At high levels of Orai opening the calcium signal co-opts myocyte voltage-dependent ion channels to provoke ~20Hz atypical automaticity and fibrillation. **2APB** causes automatic tachycardia and fibrillation in this manner.

**Figure 7. Model for Orai Arrhythmogenesis.***Left:* Under normal conditions Orais are tightly regulated and not ar‐ rhythmogenic. *Center*: Low level dysregulation (or activation) of Orais by numerous factors (*Lower right box*) will pro‐ duce a progressive arrhythmic effect. At intermediate levels the calcium signal will provoke afterdepolarization. *Right*: At high levels of Orai opening the calcium signal co-opts myocyte voltage-dependent ion channels to provoke ∼20Hz

Non-EC Ca++ store depletion ↑ SR Ca++ leak, ↑ store depletion ↑Gαq signaling @↓I<sup>K</sup> ↑ late INa ↑ ARC signaling

**2-APB**

**Ca**

**Ca**

Voltagedependent Ion channels

**Ca++ signal**

Afterdepolarization Atypical automaticity

**Orai1/3 Orai1/3 Orai1/3**

**Ca**

**Ca**

Maintenance of Ca++ stores: Stim1 regulated

**Ca**

**Ca**

102 Atrial Fibrillation - Mechanisms and Treatment

ognized arrhythmic principle (Figure 7). This requires a means to activate the Orais.

might underlie a focal mechanism for arrhythmia.

Putney reported [100] that 2-aminoethoxydiphenyl borate (2APB) activates calcium entry in non-excitable cells apparently by a store-operated mechanism. Subsequent work [101-102] conclusively demonstrated that 2APB pharmacologically opens Orai1 with an EC50 of 20μM and Orai3 with an EC50 of 13μM. 2APB also alters the ion conduction properties of these channels to enhance sodium transport. We took advantage of this Orai channel opener to in‐ terrogate in a crude manner whether activating voltage-independent calcium channels

We found that 2APB provokes a novel type of arrhythmic activity [103-106] which appears to satisfy the demands of the focal source hypothesis of Engelmann and Scherf. In particular, 2APB induces a unique atypical automaticity in non-automatic heart muscle. Specifically, 2APB provokes high frequency ectopic action potentials and muscle contraction even when added to the superfusate of quiescent left atria or left ventricular papillary muscles [105 *see Figure 3*]. That is, non-automatic heart muscle which normally requires external stimulation to produce action potentials and contract will do both spontaneously, at high-frequency, and in the absence of a triggering depolarizing stimulus if these quiescent muscles are ex‐ posed to 2APB. The action potentials produced by these spontaneously contracting muscles are identical to those produced by electrically paced untreated muscles [105 *see Figure 2 & Table I*]. That is, exposing non-automatic normal left atria and papillary muscles to >10μM 2APB causes them to produce spontaneous normal action potentials and muscle contrac‐ tions at extremely high frequency in the absence of an external electrical stimulus. Under all other conditions, these muscles require an external electrical stimulus to generate an action potential and contract (Figure 8 Left; *middle panel*). These ectopic action potentials also occur from normal resting potentials and have no visible Phase 4-type depolarization. These crite‐ ria rule that 2APB induces neither a triggered activity nor typical abnormal automaticity as defined earlier. Heart muscle thus may contain a cryptic pathway whose activation trans‐ forms non-automatic tissue to a fully automatic state.

complete SR calcium store depletion (Figure 9C). Interestingly, these muscles continue to produce spontaneous action potentials at a high rate (Figure 9D) with no discernible differ‐ ence in their characteristics compared to action potentials produced in 'calcium loaded' muscles (cp. Figures 9B & 9D). Treating these spontaneously contracting muscles with SKF-96365 abolished any residual automatic contractions (Figure 9E), and these muscle now

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**Figure 9. Ryanodine Depletion of Left Atrial SR Calcium Does Not Affect 2APB Atypical Automaticity. (A)** Me‐ chanical function of an *unpaced* left atrium treated with 300nM BayK 8644 (BayK) and 22μM 2APB (2APB). Atypical mechanical automaticity is observed. **(B)** Action potentials from an *unpaced* left atrium treated as in (A). Spontaneous, automatic action potentials are recorded. **(C)** Mechanical function of a left atrium treated as in (A), ∼8min after expo‐ sure to 800nM ryanodine (*Ryanodine*). Ryanodine depresses mechanical function by opening the ryanodine channel which leaks SR calcium (*N.B.* force scales on right of (A) & (C)). Automatic mechanical activity persists but at low levels. **(D)** Electrical activity of a left atrium treated as in (C). Spontaneous electrical activity continues unabated. **(E)** Mechani‐ cal function of an atrium treated as in (C) followed by SKF-96365 (*SKF-96365: 50*μM). SKF-96365 blocks spontaneous mechanical activity. **(F)** Electrical activity of a left atrium treated as in (E) in the absence (*Rest*) or presence (3Hz) of 3Hz pacing. No action potentials are recorded in the presence of SKF-96365; it blocks spontaneous ectopy. Pacing is re‐

We then compared how an alternate calcium efflux enhancer, caffeine affected the rate of atypical automaticity. In these experiments left atria were not 'loaded' with calcium before they were exposed to 2APB; loading was avoided to assess how the disruption of normal

quired to generate action potentials (3Hz).

calcium stores affected automaticity.

required pacing to produce action potentials (Figure 9F, *cp. Rest with 3Hz pacing*).

Several pharmacological studies assessed whether 2APB provokes atypical automaticity through the activation of Orai channels. SKF-96365, an inhibitor of calcium entry via the TRP and the Orai channels, completely prevents or reverses 2APB-linked atypical automa‐ ticity [105 *see Figure 3 & 5*]. Importantly, if paced left atria or papillary muscles are exposed to 2APB, they produce electromechanical activity independently of the pacing stimulus. That is, they produce both paced and 'spontaneous' electromechanical events. SKF-96365 added to the superfusate stops only the spontaneous ectopy which occurs independently of pacing. These isolated muscles then follow the pacing stimulus faithfully, requiring external pacing to contract or produce action potentials [105 *see e.g. Figure 5*]. ML-7, a congener of a second inhibitor of Orai-linked calcium entry [108], also suppresses this high frequency atypical automaticity as do two calmodulin inhibitors [105 *see Figure 6*]. These data suggest that the Orai channels and cell calcium signaling participate in converting non-automatic muscles to an automatic state. These atrial and papillary muscles weighed 3 to 5mg wetweight. Thus they were unlikely to support reentry based on the criteria of Garrey and Moe. The high frequency automaticity they produced following the presumed activation of the Orai channels could, however, form focal sites of paroxysmal or permanent electrical insta‐ bility in intact heart muscle.

Some current hypotheses for focal arrhythmia require high levels of SR calcium (SR calcium load) to drive afterdepolarization or more complex arrhythmia [40, 41,55]. In support of this view, increased muscle calcium has long been known to favor arrhythmogenesis. However, SR calcium load decreases in heart failure, a condition that also exhibits high rates of ar‐ rhythmia. Thus muscle calcium load appears to not always associate with ectopic activity. Despite this paradoxical complication, a great deal of exquisite experimental expertise has defined SR calcium leak rates in relation to SR calcium load with an eye toward explaining arrhythmogenic activity in failing heart [109]. Rates of 2APB atypical automaticity increase with increases in muscle calcium, so we wished to test whether SR calcium stores were, in fact, critical to the persistence of atypical automaticity. Left atria were challenged with BayK 8644 to increase their calcium content and then with 2APB to provoke high frequency atypi‐ cal automaticity. These unpaced muscles produced persistent action potentials and contrac‐ tions at a very fast rate (Figures 9A & 9B). Treating these muscles with 800nM ryanodine greatly reduces their force of contraction, evidence for ryanodine receptor opening and near complete SR calcium store depletion (Figure 9C). Interestingly, these muscles continue to produce spontaneous action potentials at a high rate (Figure 9D) with no discernible differ‐ ence in their characteristics compared to action potentials produced in 'calcium loaded' muscles (cp. Figures 9B & 9D). Treating these spontaneously contracting muscles with SKF-96365 abolished any residual automatic contractions (Figure 9E), and these muscle now required pacing to produce action potentials (Figure 9F, *cp. Rest with 3Hz pacing*).

posed to 2APB. The action potentials produced by these spontaneously contracting muscles are identical to those produced by electrically paced untreated muscles [105 *see Figure 2 & Table I*]. That is, exposing non-automatic normal left atria and papillary muscles to >10μM 2APB causes them to produce spontaneous normal action potentials and muscle contrac‐ tions at extremely high frequency in the absence of an external electrical stimulus. Under all other conditions, these muscles require an external electrical stimulus to generate an action potential and contract (Figure 8 Left; *middle panel*). These ectopic action potentials also occur from normal resting potentials and have no visible Phase 4-type depolarization. These crite‐ ria rule that 2APB induces neither a triggered activity nor typical abnormal automaticity as defined earlier. Heart muscle thus may contain a cryptic pathway whose activation trans‐

Several pharmacological studies assessed whether 2APB provokes atypical automaticity through the activation of Orai channels. SKF-96365, an inhibitor of calcium entry via the TRP and the Orai channels, completely prevents or reverses 2APB-linked atypical automa‐ ticity [105 *see Figure 3 & 5*]. Importantly, if paced left atria or papillary muscles are exposed to 2APB, they produce electromechanical activity independently of the pacing stimulus. That is, they produce both paced and 'spontaneous' electromechanical events. SKF-96365 added to the superfusate stops only the spontaneous ectopy which occurs independently of pacing. These isolated muscles then follow the pacing stimulus faithfully, requiring external pacing to contract or produce action potentials [105 *see e.g. Figure 5*]. ML-7, a congener of a second inhibitor of Orai-linked calcium entry [108], also suppresses this high frequency atypical automaticity as do two calmodulin inhibitors [105 *see Figure 6*]. These data suggest that the Orai channels and cell calcium signaling participate in converting non-automatic muscles to an automatic state. These atrial and papillary muscles weighed 3 to 5mg wetweight. Thus they were unlikely to support reentry based on the criteria of Garrey and Moe. The high frequency automaticity they produced following the presumed activation of the Orai channels could, however, form focal sites of paroxysmal or permanent electrical insta‐

Some current hypotheses for focal arrhythmia require high levels of SR calcium (SR calcium load) to drive afterdepolarization or more complex arrhythmia [40, 41,55]. In support of this view, increased muscle calcium has long been known to favor arrhythmogenesis. However, SR calcium load decreases in heart failure, a condition that also exhibits high rates of ar‐ rhythmia. Thus muscle calcium load appears to not always associate with ectopic activity. Despite this paradoxical complication, a great deal of exquisite experimental expertise has defined SR calcium leak rates in relation to SR calcium load with an eye toward explaining arrhythmogenic activity in failing heart [109]. Rates of 2APB atypical automaticity increase with increases in muscle calcium, so we wished to test whether SR calcium stores were, in fact, critical to the persistence of atypical automaticity. Left atria were challenged with BayK 8644 to increase their calcium content and then with 2APB to provoke high frequency atypi‐ cal automaticity. These unpaced muscles produced persistent action potentials and contrac‐ tions at a very fast rate (Figures 9A & 9B). Treating these muscles with 800nM ryanodine greatly reduces their force of contraction, evidence for ryanodine receptor opening and near

forms non-automatic tissue to a fully automatic state.

104 Atrial Fibrillation - Mechanisms and Treatment

bility in intact heart muscle.

**Figure 9. Ryanodine Depletion of Left Atrial SR Calcium Does Not Affect 2APB Atypical Automaticity. (A)** Me‐ chanical function of an *unpaced* left atrium treated with 300nM BayK 8644 (BayK) and 22μM 2APB (2APB). Atypical mechanical automaticity is observed. **(B)** Action potentials from an *unpaced* left atrium treated as in (A). Spontaneous, automatic action potentials are recorded. **(C)** Mechanical function of a left atrium treated as in (A), ∼8min after expo‐ sure to 800nM ryanodine (*Ryanodine*). Ryanodine depresses mechanical function by opening the ryanodine channel which leaks SR calcium (*N.B.* force scales on right of (A) & (C)). Automatic mechanical activity persists but at low levels. **(D)** Electrical activity of a left atrium treated as in (C). Spontaneous electrical activity continues unabated. **(E)** Mechani‐ cal function of an atrium treated as in (C) followed by SKF-96365 (*SKF-96365: 50*μM). SKF-96365 blocks spontaneous mechanical activity. **(F)** Electrical activity of a left atrium treated as in (E) in the absence (*Rest*) or presence (3Hz) of 3Hz pacing. No action potentials are recorded in the presence of SKF-96365; it blocks spontaneous ectopy. Pacing is re‐ quired to generate action potentials (3Hz).

We then compared how an alternate calcium efflux enhancer, caffeine affected the rate of atypical automaticity. In these experiments left atria were not 'loaded' with calcium before they were exposed to 2APB; loading was avoided to assess how the disruption of normal calcium stores affected automaticity.

Impressively, 10mM caffeine markedly increased the rate of left atrial spontaneous contrac‐ tion from 106±25 to 362±38 contractions per minute (Figure 10). This significant increase in the rate of atypical automaticity was transient as after 3 to 5 minutes of exposure to these conditions the rates of automaticity decrease to 10±7 per minute. Normal paced mechanical function remained intact albeit at lower forces of contraction because of caffeine treatment (Data not shown).

This notable result leads to four interesting speculations. First, the rates of atypical automa‐ ticity measured in caffeine-treated muscles at 30°C are about 50% faster than those meas‐ ured for 'calcium loaded' atria at the same temperature [104-105]. Thus calcium loading does not produce the most rapid rates of automaticity, caffeine a calcium efflux agent does. Second, the rate of atypical automaticity does not greatly slow with the depletion of ryano‐ dine-sensitive SR calcium. Thus a distinction must exist between ryanodine and caffeine in their interaction with the source of atypical automaticity. Third, if the rate of automatic ac‐ tivity observed with caffeine treatment exhibits identical Q10s as our earlier data (Figure 8), then this type of atypical automaticity might reach rates of ~15Hz at 37°C. Fourth, ryanodine calcium stores do not appear to greatly influence atypical automaticity indicating that this form of ectopic activity may occur readily at low and at high muscle calcium loads.

**Figure 10. Caffeine Transiently Accelerates the Rate of Atypical Automaticity.** Rat left atria (n=6) were exposed to 30μM 2APB in the absence of calcium loading agent. These muscles spontaneously contracted sporadically at a rate shown at the left of the figure. Muscles then were rapidly exposed to superfusate containing 30μM 2APB and 10mM caffeine. This treatment greatly increased the rate of atypical automatic activity to over 350 contractions per minute. After 3-5 minutes under these conditions, the rate of automaticity decreased to 10 contractions per minute.

Thus heart may possess two ways to produce action potentials that provoke contraction. First is the well-known and long-studied pathway whereby an external input derived either from the sinoatrial node or from a pacing stimulus causes myocyte depolarization. Second, an activator of the voltage-independent Orai channels appears to uncover a pathway whose activation allows non-automatic muscles to produce normal action potentials and muscle contraction independent of an external stimulus. This atypical automaticity can occur spor‐

**Figure 11. Interpretation of Ryanodine-Caffeine Effects on Atypical Automaticity.***Left.* Both ryanodine and caf‐ feine deplete SR calcium stores. *Right.* Caffeine accelerates store-operated calcium entry in some experimental set‐

**Orai**

**Ca**

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**Ca Caffeine 2APB**

Voltage-Independent Calcium Channels, Molecular Sources of Supraventricular Arrhythmia

**Orai calcium-linked arrhythmogenic signaling pathway**

**Atypical Automaticity**

**Ca\* Pool**

**Ca**

All of these experiments were performed in intact superfused muscles. Consequently, we harbored concern that conditions like hypoxia might influence our results. To address this point rat hearts were perfused in the Langendorff mode to test how 2APB affects well-oxy‐ genated muscle. These perfusions also assessed (a) whether high frequency 2APB-induced automaticity requires muscle calcium loading and (b) how 2APB affects hearts with an intact conduction system in sinus rhythm. The first point arises because the isolation of atria or

adically or at high frequency depending on the calcium loading of isolated muscles.

tings [110]. We suggest this latter effect of caffeine enhances the rates of automaticity (Figure 10).

**SR**

**SERCA**

**RyR**

**Ryanodine Caffeine**

**Ca**

**Ca**

The literature contains one possible explanation for the difference between the ryanodine and the caffeine responses observed in automatically contracting left atria. Corda noted that while ryanodine affects calcium leak from internal stores it does not activate a voltage-independent store-operated response [110]. By contrast, caffeine does. That is, in their experimental system, exposing cultured cells to caffeine provoked a prominent entry of calcium presumably through the Orai-linked store-operated channel. This result supports a contention that volt‐ age-independent calcium entry and downstream signaling are the source for atypical automa‐ ticity (Figure 11). Many more experiments are needed to establish this possibility.

Impressively, 10mM caffeine markedly increased the rate of left atrial spontaneous contrac‐ tion from 106±25 to 362±38 contractions per minute (Figure 10). This significant increase in the rate of atypical automaticity was transient as after 3 to 5 minutes of exposure to these conditions the rates of automaticity decrease to 10±7 per minute. Normal paced mechanical function remained intact albeit at lower forces of contraction because of caffeine treatment

This notable result leads to four interesting speculations. First, the rates of atypical automa‐ ticity measured in caffeine-treated muscles at 30°C are about 50% faster than those meas‐ ured for 'calcium loaded' atria at the same temperature [104-105]. Thus calcium loading does not produce the most rapid rates of automaticity, caffeine a calcium efflux agent does. Second, the rate of atypical automaticity does not greatly slow with the depletion of ryano‐ dine-sensitive SR calcium. Thus a distinction must exist between ryanodine and caffeine in their interaction with the source of atypical automaticity. Third, if the rate of automatic ac‐ tivity observed with caffeine treatment exhibits identical Q10s as our earlier data (Figure 8), then this type of atypical automaticity might reach rates of ~15Hz at 37°C. Fourth, ryanodine calcium stores do not appear to greatly influence atypical automaticity indicating that this

form of ectopic activity may occur readily at low and at high muscle calcium loads.

**Figure 10. Caffeine Transiently Accelerates the Rate of Atypical Automaticity.** Rat left atria (n=6) were exposed to 30μM 2APB in the absence of calcium loading agent. These muscles spontaneously contracted sporadically at a rate shown at the left of the figure. Muscles then were rapidly exposed to superfusate containing 30μM 2APB and 10mM caffeine. This treatment greatly increased the rate of atypical automatic activity to over 350 contractions per minute.

The literature contains one possible explanation for the difference between the ryanodine and the caffeine responses observed in automatically contracting left atria. Corda noted that while ryanodine affects calcium leak from internal stores it does not activate a voltage-independent store-operated response [110]. By contrast, caffeine does. That is, in their experimental system, exposing cultured cells to caffeine provoked a prominent entry of calcium presumably through the Orai-linked store-operated channel. This result supports a contention that volt‐ age-independent calcium entry and downstream signaling are the source for atypical automa‐

After 3-5 minutes under these conditions, the rate of automaticity decreased to 10 contractions per minute.

ticity (Figure 11). Many more experiments are needed to establish this possibility.

**106 ± 25 362 ± 38**

**10mM caffeine**

**spontaneous contractions/min** 

**5s**

(Data not shown).

106 Atrial Fibrillation - Mechanisms and Treatment

**Figure 11. Interpretation of Ryanodine-Caffeine Effects on Atypical Automaticity.***Left.* Both ryanodine and caf‐ feine deplete SR calcium stores. *Right.* Caffeine accelerates store-operated calcium entry in some experimental set‐ tings [110]. We suggest this latter effect of caffeine enhances the rates of automaticity (Figure 10).

Thus heart may possess two ways to produce action potentials that provoke contraction. First is the well-known and long-studied pathway whereby an external input derived either from the sinoatrial node or from a pacing stimulus causes myocyte depolarization. Second, an activator of the voltage-independent Orai channels appears to uncover a pathway whose activation allows non-automatic muscles to produce normal action potentials and muscle contraction independent of an external stimulus. This atypical automaticity can occur spor‐ adically or at high frequency depending on the calcium loading of isolated muscles.

All of these experiments were performed in intact superfused muscles. Consequently, we harbored concern that conditions like hypoxia might influence our results. To address this point rat hearts were perfused in the Langendorff mode to test how 2APB affects well-oxy‐ genated muscle. These perfusions also assessed (a) whether high frequency 2APB-induced automaticity requires muscle calcium loading and (b) how 2APB affects hearts with an intact conduction system in sinus rhythm. The first point arises because the isolation of atria or papillary muscles might unload unique cell calcium pools that are critical for instigating atypical automaticity. Hence the effect of the calcium loading of isolated muscles to increase the rate of 2APB automaticity [104] might be mistaken to reflect loading of the SR pool in‐ volved in contraction rather than the concurrent loading of a pool central to atypical auto‐ maticity. The second point addresses questions by Boyden and ter Keurs [88].

as a likely source of arrhythmia. If the model for the functional inter-relationship of voltageindependent calcium channels and calcium pools proposed by Shuttleworth [64], Lewis [98] and others is correct, then (a) inputs which excessively stimulate the Orai channels or (b) a yet-to-be-identified cell signaling system which mirrors the action of 2APB on the Orai chan‐ nels might create focal sources of high frequency atypical automaticity in muscle as small or smaller than 3mg wet-weight. The duration of this automaticity would depend on the pres‐

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**2-APB (μM)**

**Figure 12**: **Bcl2 Antagonists Block 2APB Atypical Automaticity**. Rat left atria (n=5 per group) were superfused and paced [105]. Atria were left untreated (**▪**) or were pre-incubated with 80μM methoxy-antimycin A3 (●) 80 μM HA-14-1 (◊), 30 μM EGCG (○), or 30 μM gossypol (x). Increasing concentrations of 2APB were added to the superfusate for 3min at each concentration. The pacing stimulus then was stopped and the rate of spontaneous atrial contraction was recorded. All four bcl2 antagonists

**Figure 12. BcI2 Antagonists Block 2APB Atypical Automaticity.** Rat left atria (n=5 per group) were superfused and paced [105]. Atria were left untreated (▪) or were pre-incubated with 80μM methoxy-antimycin A3 (∙) 80 μM HA-14-1 (◊), 30 μM EGCG (o), or 30 μM gossypol (x). Increasing concentrations of 2APB were added to the superfusate for 3min at each concentration. The pacing stimulus then was stopped and the rate of spontaneous atrial contraction was re‐

If voltage-independent calcium entry via the Orai channels initiates atrial and ventricular electrical instability *in vivo*, then this putative fourth mechanism would offer (a) a dynamic means to explain arrhythmia and (b) a mechanism which extends the three current hypothe‐ ses to explain these disorders. Dynamism arises because a multitude of physiological and pathophysiological inputs directly stimulate or indirectly activate the Orai channels [64,75,98]. The apparent arrhythmogenicity of calcium entry via the Orai channels may ex‐ tend the 'calcium leak' hypothesis for arrhythmia (Figure 5). Specifically, certain types of SR calcium leak may favor the compensatory activation of voltage-independent calcium entry [112]. Thus coupling SR calcium leak and voltage-independent calcium entry may increase the depolarizing influence which calcium leak exerts on compromised myocytes. This could contribute to the increased propensity for arrhythmia observed in 'leaky' failing hearts.

ence of input signals that stimulate voltage-independent calcium entry.

**Spontaneous Contractions(min)-1**

suppressed 2APB automaticity.

corded. All four bcI2 antagonists suppressed 2APB automaticity.

Perfused hearts yield several important and impressive results. First, the perfusion of hearts with 5μM 2APB generates spontaneous, sporadic electrical activity from multiple sites, a conclusion based on the morphology of these ectopic depolarizations [106 see Figure 2]. In‐ creasing the perfusate 2APB concentration produces striking changes in the electromechani‐ cal activity of these hearts [ http://www.dom.uab.edu/pwolkowicz/IPH\_Fibrillation2- APB.mov ]. After a few minutes of perfusion with 22μM 2APB hearts begin to produce spontaneous but broad QRS complexes and contract at rates that increase from sinus rhythm (~5Hz) to upwards of 12Hz [106 *see Figures 3 & 4*]. Heart mechanical function briefly follows this ectopic electrical activity but electro-mechanical dissociation occurs as ectopic electrical activity continues to steadily increase in rate to apparent values of ~20Hz. These hearts lose the ability to generate organized mechanical activity which may reflect a form of fibrillatory conduction originating from high frequency focal sources [25,111]. A brief but impressive in‐ crease in diastolic pressure from 5mm to ~60mmHg accompanies electromechanical dissoci‐ ation. A minute or so later fibrillating hearts reach and then maintain a resting tension of 30mmHg [106 *see Figures 3 & 4*]. Coronary flow remains normal during fibrillation and per‐ sistent contracture lessening the possibility that ischemia occurs in these preparations.

The Orai and TRP channel inhibitor SKF-96365 reverses this ventricular fibrillation in an in‐ triguing way [106 *see Figure 5*] [ http://www.dom.uab.edu/pwolkowicz/IPH\_SKF-96365-Re‐ versal.mov ]. After a few minutes of perfusion with 2APB and 20μM SKF-96365, the electrical disorganization recorded by our bipolar electrodes begins to resolve. The resolu‐ tion of electrical fibrillation occurs quickly over about one second but at first it is only a tran‐ sient event as electrical instability reappears immediately after this initial flash of stability. As the time of perfusion with SKF-96365 increases, periods of stable electrical activity get longer until sinus rhythm is restored. In all cases, the temporal interface between fibrillation and electrical quiescence produce a rapid decrease in resting tension from 30mmm to about 8mmHg, mechanical quiescence, and then normal, potentiated mechanical contractions [106 *see Figure 5*]. This very rapid restoration of normal diastolic pressure and electro-mechanical quiescence most likely are not caused by a decrease in bulk cytosolic calcium throughout the heart. We suspect this change likely reflects the interdiction by SKF-96365 of signaling events initiated by 2APB. Note that 2APB remains in the perfusate throughout these experi‐ ments. These electrical results remarkably mimic those reported by Hirose for Gαq overex‐ pressing mice [96]. Together these data indicate that SKF-96365 suppresses fibrillation caused by genetic enhancement of the initiation site for voltage-independent calcium signal‐ ing [96], the Gαq receptor, and by perfusion with a pharmacological activator of Orai chan‐ nel calcium entry, which might occur in Gαq transgenic mice for reasons stated earlier. The concentrations of 2APB used in our work inhibit both the IP3Rs and many of the relevant TRPC channels. Thus the voltage-independent Orai channels, either Orai1 or Orai3, appear as a likely source of arrhythmia. If the model for the functional inter-relationship of voltageindependent calcium channels and calcium pools proposed by Shuttleworth [64], Lewis [98] and others is correct, then (a) inputs which excessively stimulate the Orai channels or (b) a yet-to-be-identified cell signaling system which mirrors the action of 2APB on the Orai chan‐ nels might create focal sources of high frequency atypical automaticity in muscle as small or smaller than 3mg wet-weight. The duration of this automaticity would depend on the pres‐ ence of input signals that stimulate voltage-independent calcium entry.

papillary muscles might unload unique cell calcium pools that are critical for instigating atypical automaticity. Hence the effect of the calcium loading of isolated muscles to increase the rate of 2APB automaticity [104] might be mistaken to reflect loading of the SR pool in‐ volved in contraction rather than the concurrent loading of a pool central to atypical auto‐

Perfused hearts yield several important and impressive results. First, the perfusion of hearts with 5μM 2APB generates spontaneous, sporadic electrical activity from multiple sites, a conclusion based on the morphology of these ectopic depolarizations [106 see Figure 2]. In‐ creasing the perfusate 2APB concentration produces striking changes in the electromechani‐ cal activity of these hearts [ http://www.dom.uab.edu/pwolkowicz/IPH\_Fibrillation2- APB.mov ]. After a few minutes of perfusion with 22μM 2APB hearts begin to produce spontaneous but broad QRS complexes and contract at rates that increase from sinus rhythm (~5Hz) to upwards of 12Hz [106 *see Figures 3 & 4*]. Heart mechanical function briefly follows this ectopic electrical activity but electro-mechanical dissociation occurs as ectopic electrical activity continues to steadily increase in rate to apparent values of ~20Hz. These hearts lose the ability to generate organized mechanical activity which may reflect a form of fibrillatory conduction originating from high frequency focal sources [25,111]. A brief but impressive in‐ crease in diastolic pressure from 5mm to ~60mmHg accompanies electromechanical dissoci‐ ation. A minute or so later fibrillating hearts reach and then maintain a resting tension of 30mmHg [106 *see Figures 3 & 4*]. Coronary flow remains normal during fibrillation and per‐ sistent contracture lessening the possibility that ischemia occurs in these preparations.

The Orai and TRP channel inhibitor SKF-96365 reverses this ventricular fibrillation in an in‐ triguing way [106 *see Figure 5*] [ http://www.dom.uab.edu/pwolkowicz/IPH\_SKF-96365-Re‐ versal.mov ]. After a few minutes of perfusion with 2APB and 20μM SKF-96365, the electrical disorganization recorded by our bipolar electrodes begins to resolve. The resolu‐ tion of electrical fibrillation occurs quickly over about one second but at first it is only a tran‐ sient event as electrical instability reappears immediately after this initial flash of stability. As the time of perfusion with SKF-96365 increases, periods of stable electrical activity get longer until sinus rhythm is restored. In all cases, the temporal interface between fibrillation and electrical quiescence produce a rapid decrease in resting tension from 30mmm to about 8mmHg, mechanical quiescence, and then normal, potentiated mechanical contractions [106 *see Figure 5*]. This very rapid restoration of normal diastolic pressure and electro-mechanical quiescence most likely are not caused by a decrease in bulk cytosolic calcium throughout the heart. We suspect this change likely reflects the interdiction by SKF-96365 of signaling events initiated by 2APB. Note that 2APB remains in the perfusate throughout these experi‐ ments. These electrical results remarkably mimic those reported by Hirose for Gαq overex‐ pressing mice [96]. Together these data indicate that SKF-96365 suppresses fibrillation caused by genetic enhancement of the initiation site for voltage-independent calcium signal‐ ing [96], the Gαq receptor, and by perfusion with a pharmacological activator of Orai chan‐ nel calcium entry, which might occur in Gαq transgenic mice for reasons stated earlier. The concentrations of 2APB used in our work inhibit both the IP3Rs and many of the relevant TRPC channels. Thus the voltage-independent Orai channels, either Orai1 or Orai3, appear

maticity. The second point addresses questions by Boyden and ter Keurs [88].

108 Atrial Fibrillation - Mechanisms and Treatment

**Figure 12**: **Bcl2 Antagonists Block 2APB Atypical Automaticity**. Rat left atria (n=5 per group) were superfused and paced [105]. Atria were left untreated (**▪**) or were pre-incubated with 80μM methoxy-antimycin A3 (●) 80 μM HA-14-1 (◊), 30 μM EGCG (○), or 30 μM gossypol (x). Increasing concentrations of 2APB were added to the superfusate for 3min at each concentration. The pacing stimulus then was stopped and the rate of **Figure 12. BcI2 Antagonists Block 2APB Atypical Automaticity.** Rat left atria (n=5 per group) were superfused and paced [105]. Atria were left untreated (▪) or were pre-incubated with 80μM methoxy-antimycin A3 (∙) 80 μM HA-14-1 (◊), 30 μM EGCG (o), or 30 μM gossypol (x). Increasing concentrations of 2APB were added to the superfusate for 3min at each concentration. The pacing stimulus then was stopped and the rate of spontaneous atrial contraction was re‐ corded. All four bcI2 antagonists suppressed 2APB automaticity.

spontaneous atrial contraction was recorded. All four bcl2 antagonists suppressed 2APB automaticity. If voltage-independent calcium entry via the Orai channels initiates atrial and ventricular electrical instability *in vivo*, then this putative fourth mechanism would offer (a) a dynamic means to explain arrhythmia and (b) a mechanism which extends the three current hypothe‐ ses to explain these disorders. Dynamism arises because a multitude of physiological and pathophysiological inputs directly stimulate or indirectly activate the Orai channels [64,75,98]. The apparent arrhythmogenicity of calcium entry via the Orai channels may ex‐ tend the 'calcium leak' hypothesis for arrhythmia (Figure 5). Specifically, certain types of SR calcium leak may favor the compensatory activation of voltage-independent calcium entry [112]. Thus coupling SR calcium leak and voltage-independent calcium entry may increase the depolarizing influence which calcium leak exerts on compromised myocytes. This could contribute to the increased propensity for arrhythmia observed in 'leaky' failing hearts. With respect to 'abnormal' automaticity, exuberant Orai channel activation might produce automatic foci under conditions that do not require partial myocyte depolarization. Regard‐ ing reentry, if dysregulated Orai channels were *in vivo* sources of >10Hz atypical automatici‐ ty, then they could lead to ectopy at rates that promote atrial (*and ventricular*) fibrillatory conduction which begins at 6Hz [111]. Finally, if this fourth mechanism of voltage-inde‐ pendent arrhythmogenesis were to hold in Purkinje cells, it might explain some types of idi‐ opathic ventricular fibrillation [113].

We are cognizant that the available data allow for only the barest of frameworks for the hy‐ pothesis that voltage-independent calcium entry and signaling are important sources of fo‐ cal arrhythmia. Undoubtedly multiple unexpected cellular signaling intermediates participate in this pathway and they may provide new targets for anti-arrhythmics. For ex‐ ample, bcl-2 is a small protein that regulates the intrinsic pathway for apoptosis through its interaction with mitochondria. By contrast, Distelhorst [114] has championed the concept that bcl-2 binding to the IP3R suppresses calcium signals related to apoptosis while enhanc‐ ing signals related to cell survival. Sub-sets of bcl-2 bind to the endoplasmic reticulum and the plasma membrane [115]. Thus bcl-2 might play a role in the atypical automaticity which 2APB induces. To test this possibility rat left atria were superfused and paced at 0.1Hz as previously described [105]. Left atria (n=5 per group) were then left untreated or were pretreated with 80μM HA14-1 and methoxyantimycin A, two cell permeable inhibitors of bcl-2, or with 30μM of two naturally occurring bcl-2 inhibitors EGCG and gossypol [116-119]. In‐ creasing concentrations of 2APB from 0 to 30μM were added to the superfusate and the rate of spontaneous activity was recorded after three minute incubation at any concentration. All four bcl-2 inhibitors significantly or completely prevented atypical automatic activity (Fig‐ ure 12). EGCG and gossypol were tested for their ability to reverse high frequency atypical automaticity in left atria treated with BayK 8644 and 20μM 2APB. Both naturally occurring bcl-2 inhibitors reversed this automatic activity but did not affect normal paced muscle con‐ traction (Figure 13). Thus bcl-2 may play a role in this type of ectopy but these provocative data require significant follow-on experiments to more firmly establish this conclusion.

The evidence summarized in this review suggests the existence of a cell mechanism to gen‐ erate focal arrhythmia. Some limitations must be addressed to afford a more sound footing for the concept that focal, cellular sources of arrhythmia arise from the activation of voltageindependent calcium channels.

lished under separate auspices which demonstrate that these signaling events profoundly change the fundamental characteristics of heart voltage-dependent ion channels. We be‐ lieve these fundamental changes underlie the fourth mechanism for arrhythmia we pro‐

staunch atypical automaticity. Gossypol induced a 50% reduction at 10μM while EGCG required 25μM.

**Figure 13. Naturally Occurring bcl2 Antagonists Suppress 2APB Atypical Automaticity.***Left.* Rat let atrium was su‐ perfused, paced at 0.1Hz, and treated with 300nM BayK 8644 [105]. 2APB (22μM) was added where indicated (2APB). After two to three minutes this atrium began to spontaneously contract at a high rate (*dark area in center of trace*). The pacing stimulus then was stopped. (*Rest*). Atypical automatic activity persists unabated. EGCG (30μM) was added where indicated (EGCG). Shortly thereafter, atypical automaticity ceased and this *now* **unpaced** muscle became quies‐ cent. The 0.1Hz pacing stimulus then was reinstated and the left atrium faithfully followed it (*Right 0.1Hz*). *Right.* Aver‐ age of exeriments like the one shown to the left where increasing concentrations of gossypol and EGCG were used to

**Spontaneous Contractions (% Initial)**

Voltage-Independent Calcium Channels, Molecular Sources of Supraventricular Arrhythmia

**0 10 20 30**

**Gossypol (■) EGCG (○) (μM)**

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**•** Can this voltage-independent mechanism provide a connection between afterdepolariza‐ tion and sustained triggered activity/high frequency atypical automaticity? To test this possibility we investigated whether voltage-independent calcium channels also partici‐ pate in the triggered activity that occurs with increased late sodium current. We find that atria treated with appropriate concentrations of sea anemone toxin type II [33] produce triggered early afterdepolarization in a steady-state manner. Our preliminary data to be published elsewhere show that (a) the ARC channel inhibitor LOE-908, (b) an antibody that binds plasma membrane Stim1 which is required for ARC channel activity, (c) the Orai inhibitor SKF-96365, (d) the CaMKII inhibitor KN-93, and (e) several inhibitors of the calcium-dependent cytosolic phospholipase A2 all suppress late sodium current-induced triggered activity. Importantly, we assessed whether SKF-96365 shortens action potential duration in atria treated with sea anemone toxin. It does not. Also neither LOE-908 nor

Thus related panels of voltage-independent calcium signaling inhibitors suppress early af‐ terdepolarization and/or 2APB automaticity. We propose that the calcium loading which oc‐

KN-93 suppresses the high frequency automaticity which 2APB induces.

pose here.


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With respect to 'abnormal' automaticity, exuberant Orai channel activation might produce automatic foci under conditions that do not require partial myocyte depolarization. Regard‐ ing reentry, if dysregulated Orai channels were *in vivo* sources of >10Hz atypical automatici‐ ty, then they could lead to ectopy at rates that promote atrial (*and ventricular*) fibrillatory conduction which begins at 6Hz [111]. Finally, if this fourth mechanism of voltage-inde‐ pendent arrhythmogenesis were to hold in Purkinje cells, it might explain some types of idi‐

We are cognizant that the available data allow for only the barest of frameworks for the hy‐ pothesis that voltage-independent calcium entry and signaling are important sources of fo‐ cal arrhythmia. Undoubtedly multiple unexpected cellular signaling intermediates participate in this pathway and they may provide new targets for anti-arrhythmics. For ex‐ ample, bcl-2 is a small protein that regulates the intrinsic pathway for apoptosis through its interaction with mitochondria. By contrast, Distelhorst [114] has championed the concept that bcl-2 binding to the IP3R suppresses calcium signals related to apoptosis while enhanc‐ ing signals related to cell survival. Sub-sets of bcl-2 bind to the endoplasmic reticulum and the plasma membrane [115]. Thus bcl-2 might play a role in the atypical automaticity which 2APB induces. To test this possibility rat left atria were superfused and paced at 0.1Hz as previously described [105]. Left atria (n=5 per group) were then left untreated or were pretreated with 80μM HA14-1 and methoxyantimycin A, two cell permeable inhibitors of bcl-2, or with 30μM of two naturally occurring bcl-2 inhibitors EGCG and gossypol [116-119]. In‐ creasing concentrations of 2APB from 0 to 30μM were added to the superfusate and the rate of spontaneous activity was recorded after three minute incubation at any concentration. All four bcl-2 inhibitors significantly or completely prevented atypical automatic activity (Fig‐ ure 12). EGCG and gossypol were tested for their ability to reverse high frequency atypical automaticity in left atria treated with BayK 8644 and 20μM 2APB. Both naturally occurring bcl-2 inhibitors reversed this automatic activity but did not affect normal paced muscle con‐ traction (Figure 13). Thus bcl-2 may play a role in this type of ectopy but these provocative data require significant follow-on experiments to more firmly establish this conclusion.

The evidence summarized in this review suggests the existence of a cell mechanism to gen‐ erate focal arrhythmia. Some limitations must be addressed to afford a more sound footing for the concept that focal, cellular sources of arrhythmia arise from the activation of voltage-

**•** How does the activation of voltage-independent calcium channels produce electrome‐ chanical instability in intact heart muscles (Figure 7, Calcium signal)? Does voltage-inde‐ pendent calcium entry *per se*, that is calcium acting as a charge carrier, activate this high frequency ectopy? Or, do calcium signaling events specific to this type of channel lead non-automatic heart muscle to become automatic? We favor the latter view. Supporting this possibility calmodulin inhibitors suppress atypical automaticity in heart muscles treated with 2APB [105]. By contrast CaMKII inhibitors do not [105]. Thus calmodulin tar‐

**•** What molecular entities lie between voltage-independent calcium signaling and automat‐ ic depolarization at rates of 10-12Hz? While a current view would favor calcium ions themselves as the arrhythmogenic principle, we have preliminary data that will be pub‐

gets other than CaMKII may be involved in this automatic activity.

opathic ventricular fibrillation [113].

110 Atrial Fibrillation - Mechanisms and Treatment

independent calcium channels.

**Figure 13. Naturally Occurring bcl2 Antagonists Suppress 2APB Atypical Automaticity.***Left.* Rat let atrium was su‐ perfused, paced at 0.1Hz, and treated with 300nM BayK 8644 [105]. 2APB (22μM) was added where indicated (2APB). After two to three minutes this atrium began to spontaneously contract at a high rate (*dark area in center of trace*). The pacing stimulus then was stopped. (*Rest*). Atypical automatic activity persists unabated. EGCG (30μM) was added where indicated (EGCG). Shortly thereafter, atypical automaticity ceased and this *now* **unpaced** muscle became quies‐ cent. The 0.1Hz pacing stimulus then was reinstated and the left atrium faithfully followed it (*Right 0.1Hz*). *Right.* Aver‐ age of exeriments like the one shown to the left where increasing concentrations of gossypol and EGCG were used to staunch atypical automaticity. Gossypol induced a 50% reduction at 10μM while EGCG required 25μM.

lished under separate auspices which demonstrate that these signaling events profoundly change the fundamental characteristics of heart voltage-dependent ion channels. We be‐ lieve these fundamental changes underlie the fourth mechanism for arrhythmia we pro‐ pose here.

**•** Can this voltage-independent mechanism provide a connection between afterdepolariza‐ tion and sustained triggered activity/high frequency atypical automaticity? To test this possibility we investigated whether voltage-independent calcium channels also partici‐ pate in the triggered activity that occurs with increased late sodium current. We find that atria treated with appropriate concentrations of sea anemone toxin type II [33] produce triggered early afterdepolarization in a steady-state manner. Our preliminary data to be published elsewhere show that (a) the ARC channel inhibitor LOE-908, (b) an antibody that binds plasma membrane Stim1 which is required for ARC channel activity, (c) the Orai inhibitor SKF-96365, (d) the CaMKII inhibitor KN-93, and (e) several inhibitors of the calcium-dependent cytosolic phospholipase A2 all suppress late sodium current-induced triggered activity. Importantly, we assessed whether SKF-96365 shortens action potential duration in atria treated with sea anemone toxin. It does not. Also neither LOE-908 nor KN-93 suppresses the high frequency automaticity which 2APB induces.

Thus related panels of voltage-independent calcium signaling inhibitors suppress early af‐ terdepolarization and/or 2APB automaticity. We propose that the calcium loading which oc‐ curs with increased late sodium current stimulates cytosolic phospholipase A2 to produce the arachidonate ligand for ARC (Figure 14). Calcium entry through the voltage-independ‐ ent ARC calcium channel may activate CaMKII which participates in provoking early after‐ depolarization. Conditions in which calcium stores begin to deplete would stimulate voltage-independent calcium entry through Orai1 [64]. Greater store depletion resulting from exuberant ARC channel activity would lead to fulminant voltage-independent calcium entry through the Orai1 calcium channel [64] and possibly a 'sustained triggered activity' which resembles 2APB-linked automaticity.

Are there intracellular, naturally occurring activators of Orai1 or Orai3 that mimic 2APB? At present none are known but such Orai-activators would link cell signaling with focal ar‐ rhythmogenesis. The interrelationship between ARC and store-operated voltage-independ‐ ent calcium entry [64] may be one such link.

The following four figures outline a putative mechanism through which voltage-independ‐ ent calcium signaling might induce afterdepolarization, sporadic and high-frequency atypi‐ cal automaticity.

**Figure 15. A Putative Mechanism for Focal Ectopy.***Left.* In excitable cells, voltage-independent & voltage-depend‐ ent ion channels co-exist. Both sets of channels respond to common or distinct inputs to produce (a) calcium signals for cell homeostasis and (b) action potentials. These two events are generally considered as separate and non-interact‐ ing entities [22,25,55,64,75,76]. *Right.* A wide range of systemic, myocyte, and local inputs may provoke the 'inappro‐ priate' interaction of these two systems. This results in voltage-independent signaling events co-opting myocyte voltage-dependent ion channels. Myocytes transform from a non-automatic to an automatic state. This change can occur for an inconstant time period and it is reversible. AA=arachidonate; LK=leukotrienes; PG=prostaglandins

Voltage-Independent Calcium Channels, Molecular Sources of Supraventricular Arrhythmia

**Orai Orai Orai NCX**

**Ca 3Na**

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113

**++**

**+ Ca 3Na+**

**NCX-coupled inward current**

**Ca Ca/Na Ca**

**regulated activity Dysregulated cardiac Orai activity**

**Ca Ca/Na Ca**

**Figure 16. Potential Arrhythmogenic Mechanisms for Dysfunctional Orai Channels.** Our data suggest dysfunc‐ tional Orais may be a source of focal arrhythmia. *Left.* These proteins normally are well-regulated by the Stim1 calcium sensing system & faithfully fulfill their function of refilling cell calcium stores. *Second from left.* Dysregulated Orals will enhance calcium transport which might activate a cryptic arrhychmogenic calcium signaling pathway. **We favor this view.** *Second from Right & Right*. Dysregulated Oral calcium transport may generate a persistent inward current which acts independent of changes in membrane potential associated with the action potential or Orai calcium entry may couple with the sodium-calcium exchanger (NCX) to create a futile calcium cycle that drives myocyte depolarization.

Such a general type of mechanism is similar to that proposed by Huo to explain 2APB ectopy [120].

**'Persistent' inward current**

**Arrhythmogenic calcium signal**

**Inactive or Stim1**

**Ca**

**Orai**

**Ca**

**Figure 14**: **Model Linking Voltage-Independent ARC Channel and Late Sodium Current Early Afterdepolarization**. *Left*. Late sodium current loads myocytes first with sodium and then wit calcium [33]. This loading activates cytosolic phospholipase A2 (cPLA2) to hydrolyze membrane phospholipids and release free arachidonate. *Right*. This eicosanoid activates the Orai1/Orai pentamer ARC channel which permits calcium entry into myocytes. This also disturbs intracellula calcium stores [64]. CaMKII may participate in this pathway by phosphorylating cPLA2 o **Figure 14. Model linking voltage-independent ARC channel and late sodium current early afterdepolariza‐ tion.***Left.* Late sodium current loads myocytes first with sodium and then with calcium [33]. This loading activates cyto‐ solic phospholipase A2 (cPLA2) to hydrolyze membrane phospholipids and release free arachidonate. *Right*. This eicosonoid activates the Orail/Orai3 pentamer ARC channel which permits calcium entry into myocytes. This also dis‐ turbs intracellular calcium stores [64]. CaMKII may participate in this pathway by phosphorylating cPLA2 or elsewhere. Low level ARC activity leads to afterdepolarization. More intense ARC activity depletes myocyte calcium stores, opens Orais and provokes high frequency sustained triggered activity. Both events require the co-opting of cardiac voltagedependent ion channels to produce spontaneous depolarization.

Voltage-Independent Calcium Channels, Molecular Sources of Supraventricular Arrhythmia http://dx.doi.org/10.5772/53649 113

curs with increased late sodium current stimulates cytosolic phospholipase A2 to produce the arachidonate ligand for ARC (Figure 14). Calcium entry through the voltage-independ‐ ent ARC calcium channel may activate CaMKII which participates in provoking early after‐ depolarization. Conditions in which calcium stores begin to deplete would stimulate voltage-independent calcium entry through Orai1 [64]. Greater store depletion resulting from exuberant ARC channel activity would lead to fulminant voltage-independent calcium entry through the Orai1 calcium channel [64] and possibly a 'sustained triggered activity'

Are there intracellular, naturally occurring activators of Orai1 or Orai3 that mimic 2APB? At present none are known but such Orai-activators would link cell signaling with focal ar‐ rhythmogenesis. The interrelationship between ARC and store-operated voltage-independ‐

The following four figures outline a putative mechanism through which voltage-independ‐ ent calcium signaling might induce afterdepolarization, sporadic and high-frequency atypi‐

**Orai3 Orai1**

**Ca**

**CaMKII?**

**Figure 14**: **Model Linking Voltage-Independent ARC Channel and Late Sodium Current Early Afterdepolarization**. *Left*. Late sodium current loads myocytes first with sodium and then wit calcium [33]. This loading activates cytosolic phospholipase A2 (cPLA2) to hydrolyze membrane phospholipids and release free arachidonate. *Right*. This eicosanoid activates the Orai1/Orai pentamer ARC channel which permits calcium entry into myocytes. This also disturbs intracellula calcium stores [64]. CaMKII may participate in this pathway by phosphorylating cPLA2 o

**Figure 14. Model linking voltage-independent ARC channel and late sodium current early afterdepolariza‐ tion.***Left.* Late sodium current loads myocytes first with sodium and then with calcium [33]. This loading activates cyto‐ solic phospholipase A2 (cPLA2) to hydrolyze membrane phospholipids and release free arachidonate. *Right*. This eicosonoid activates the Orail/Orai3 pentamer ARC channel which permits calcium entry into myocytes. This also dis‐ turbs intracellular calcium stores [64]. CaMKII may participate in this pathway by phosphorylating cPLA2 or elsewhere. Low level ARC activity leads to afterdepolarization. More intense ARC activity depletes myocyte calcium stores, opens Orais and provokes high frequency sustained triggered activity. Both events require the co-opting of cardiac voltage-

**Afterdepolarization**

**Arrhythmogenic signaling pathway**

**Orai1 Orai3**

**Ca**

**Sustained ectopy**

**Ca**

**Ca**

which resembles 2APB-linked automaticity.

112 Atrial Fibrillation - Mechanisms and Treatment

ent calcium entry [64] may be one such link.

**cPLA2**

dependent ion channels to produce spontaneous depolarization.

**cPLA2**

**CaMKII? CaMKII?**

cal automaticity.

**↑ late INa ↓I<sup>K</sup> & ↑Gαq**

**↑ Ca**

**Phospholipid**

**Arachidonate**

**Lysophospholipid**

**Figure 15. A Putative Mechanism for Focal Ectopy.***Left.* In excitable cells, voltage-independent & voltage-depend‐ ent ion channels co-exist. Both sets of channels respond to common or distinct inputs to produce (a) calcium signals for cell homeostasis and (b) action potentials. These two events are generally considered as separate and non-interact‐ ing entities [22,25,55,64,75,76]. *Right.* A wide range of systemic, myocyte, and local inputs may provoke the 'inappro‐ priate' interaction of these two systems. This results in voltage-independent signaling events co-opting myocyte voltage-dependent ion channels. Myocytes transform from a non-automatic to an automatic state. This change can occur for an inconstant time period and it is reversible. AA=arachidonate; LK=leukotrienes; PG=prostaglandins

**Figure 16. Potential Arrhythmogenic Mechanisms for Dysfunctional Orai Channels.** Our data suggest dysfunc‐ tional Orais may be a source of focal arrhythmia. *Left.* These proteins normally are well-regulated by the Stim1 calcium sensing system & faithfully fulfill their function of refilling cell calcium stores. *Second from left.* Dysregulated Orals will enhance calcium transport which might activate a cryptic arrhychmogenic calcium signaling pathway. **We favor this view.** *Second from Right & Right*. Dysregulated Oral calcium transport may generate a persistent inward current which acts independent of changes in membrane potential associated with the action potential or Orai calcium entry may couple with the sodium-calcium exchanger (NCX) to create a futile calcium cycle that drives myocyte depolarization. Such a general type of mechanism is similar to that proposed by Huo to explain 2APB ectopy [120].

the importance of voltage-independent cell signaling in heart ectopy. That is, they assume that voltage-independent cell signaling does not influence cardiac voltage-dependent ion channels or affect arrhythmia. One corollary of this dominant electrocentric view of arrhyth‐ mia is that the fundamental biophysical properties of voltage-dependent ion channels meas‐ ured in normal muscle are the only properties these proteins can evince, that these ion channels are essentially immutable activities. However, clinical and experimental data sug‐ gest that one or more signaling events greatly influence the electrical properties of heart

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Our data demonstrate that a recognized activator of the voltage-independent Orai calcium channels provokes persistent or paroxysmal tachycardia at rates of up to 12Hz in non-auto‐ matic rat left atrial or left ventricular papillary muscles. This activator also induces a reversi‐ ble type of fibrillation in intact perfused rat hearts. These data lead to the hypothesis outlined here that calcium entry through voltage-independent ion channels, specifically through the Orai channels, and/or calcium signaling events downstream of these channels elicit ectopic electrical activity in atrial (*and ventricular*) muscle. This hypothesis implies that a wide range of extracellular and intracellular signals may disrupt heart muscle electrical stability through their actions on voltage-independent calcium homeostasis that enhance voltage-independent calcium channel activity. This hypothesis provides a framework for fu‐ ture experimental tests of whether voltage-independent calcium signaling related to auto‐ nomic activity, to stress or to calcium store filling state are key molecular sources for arrhythmia. Importantly, our data to be published elsewhere indicate that dysregulated voltage-independent calcium signaling alter the fundamental characteristics of voltage-de‐ pendent ion channels, transforming them from non-automatic activities that require an ex‐ ternal depolarizing influence to automatic activities that spontaneously depolarize heart muscle. If rigorously validated, this fourth putative arrhythmogenic mechanism would sat‐

The authors gratefully acknowledge the help of Dr. Pei Pei Wang in conducting the per‐ fused heart studies described here. PEW appreciatively thanks Dr. David D. Ku of the UAB Department of Pharmacology for unfettered access to lab space and equipment during the course of much of this work. PEW thanks Dr. Jian Huang of the UAB Department of Medi‐ cine for performing microelectrode measurements of left atrial action potential mentioned in the text. He also gratefully acknowledges the assistance of Drs. Hernan E. Grenett, Edlue Ta‐ bengwa, and John C. Chatham of UAB at various points along this journey. He also would like to thank Dr. Neal Kay of UAB for his interest in this endeavor. PEW is the President and Chief Scientific Officer of KOR Therapies, LLC which seeks to develop the technology dis‐ cussed in this paper. This technology is now controlled by the University of Alabama Re‐

muscle and somehow increase its ability to generate ectopic electrical impulses.

isfy the 'focal source' hypothesis for arrhythmia.

search Foundation and under patent review.

**Acknowledgements**

**Figure <sup>17</sup>**: **Potential Cellular Mechanism for Orai Atypical Automaticity**. Our preliminary data favor <sup>a</sup> mechanism for Orai atypical automaticity in which calcium entry activates a signaling pathway that modifies the properties of the voltage-dependent ion channels critical to myocyte excitation. We do not have a **Figure 17. Potential Cellular Mechanism for Orai Atypical Automaticity.** Our preliminary data favor a mechanism for Orai atypical automaticity in which calcium entry activates a signaling pathway that modifies the properties of the voltage-dependent ion channels critical to myocyte excitation. We do not have a preferred view as to how this modifi‐ cation occurs but the lower open box notes four possibilities we are now investigating

preferred view as to how this modification occurs but the lower open box notes
