**8. Mechanism 4: Relevant overview of voltage-independent calcium homeostasis**

tracellular calcium homeostasis. Furthermore, the spontaneous action potentials these 'fail‐ ing' cells produced showed no Phase 4 depolarization which might occur if cell funny currents had somehow become active. One interpretation of this provocative report is that pathological conditions like failure change the fundamental properties of ventricular myo‐ cytes, transforming normal, non-automatic myocytes into cells that are capable of an atypi‐ cal automatic activity. This change survives cell isolation indicating it is reasonably permanent and possibly acutely reversible. Nuss did not define a mechanism to transform non-automatic (*normal*) myocytes to sporadically or rapidly automatic (*failing*) ones. The voltage-independent arrhythmogenic pathway we describe in some detail below is one can‐

Robichaux and others [68] assessed the arrhythmogenic mechanisms that underlie experi‐ mental fibrillation and reported that reentry does not predominant either soon after the in‐ duction of faradic fibrillation or several minutes after the start of fibrillation. Rather they showed that organized sources of relatively regular high frequency ectopic activity drives long-duration ventricular fibrillation. Others also have reported that focal sources of non-re‐ entrant activity predominate during experimental ventricular fibrillation [69]. Automatic ac‐ tivity was among the proposed explanations for both sets of data. It is possible that an atypical form of automaticity underlies these results and affords an unrecognized means to

The three current mechanisms for arrhythmia assume that abnormalities in (a) the well-de‐ fined process of normal cardiac excitation-contraction coupling, (b) the propagation of elec‐ trical waves through the heart or (c) the electrical response of heart muscle to enormous faradic insults circumscribe all the properties of the myocardium needed to fully explain clinical arrhythmia including atrial fibrillation. In other words, all other non-electrical cell processes are by-standers in arrhythmogenesis and they little influence heart muscle electri‐ cal stability. None of these three theories is a true focal hypothesis for arrhythmia as envi‐

Our fourth view of arrhythmia proposes that non-electrical cell signaling events can destabi‐ lize the electrical activity of myocytes and conduction system cells. Such destabilization pro‐ duces isolated focal ectopic events or high frequency focal tachycardia or fibrillation. Thus our unconventional hypothesis for arrhythmia proposes that heart muscle can produce elec‐ tromechanical activity in two ways. First is by the well-defined pathway of sinus rhythm and impulse conduction. This pathway for normal heart electromechanical activity integra‐ tes heart function with systemic physiology. Second, the activation of a cellular 'arrhythmo‐ genic' signaling pathway can transform non-automatic myocytes into cells that spontaneously produce sporadic or high frequency electrical activity independent of exter‐ nal regulators like sinus rhythm or systemic physiology. Aberrant or exuberant myocyte or Purkinje cell voltage-independent calcium homeostasis is one means we have identified to

produce sporadic or high frequency myocardial electrical instability.

**7. Summary of the mechanisms for arrhythmia**

sioned by Engelman and championed by Scherf and others.

didate mechanism.

92 Atrial Fibrillation - Mechanisms and Treatment

Cell calcium entry and cell calcium homeostasis are divided operationally into voltage-inde‐ pendent and voltage-dependent domains. Voltage-independent calcium homeostasis regu‐ lates non-excitable and excitable cell signaling events that are critical to cell growth, survival, and death. Four families of proteins control the generation and propagation of these calcium signals thereby allowing cells to respond appropriately to challenges or changes in their environment. Two families of plasma membrane calcium transporters per‐ mit voltage-independent calcium entry in response to extra- or intra-cellular signals. While cell membrane potential influences these carriers, they are not voltage-gated proteins. A third family of intracellular calcium release channels interacts functionally with these trans‐ porters. A fourth family maintains the cell calcium stores used to continually generate calci‐ um signals, a task critical for cell viability. None of these families of voltage-independent proteins is now widely believed to greatly influence heart excitability. Our data and that of others directly challenge this view. They propose that deranged voltage-independent calci‐ um homeostasis and we suggest the dysregulated activity of one family of voltage-inde‐ pendent calcium channels can provoke heart muscle electrical instability.

The first family is the well-characterized Gαq-coupled receptor proteins (Figure 4). A broad range of agonists including bioactive peptides like angiotensin II, bioactive lipids like pros‐ taglandins, and hormones like norepinephrine stimulate this family of receptors. Agonist binding to a specific Gαq-coupled receptor activates a plasma membrane phosphatidylinosi‐ toyl-specific phospholipase C. This lipase generates two active intermediates for voltage-in‐ dependent calcium signaling. Water-soluble inositol-1,4,5-trisphosphate is the first intermediate as defined in the elegant work of Berridge in the 1970s [70]. The second is the membrane-bound lipid diacylglycerol. A highly complex interaction among G-protein regu‐ lators, inositol phosphate kinases and phosphatases, and diacylglycerol kinases and lipases set the rate of production and the steady-state levels of these signaling intermediates.

Inositol-1,4,5-trisphosphate diffuses from the environ of the cytosolic face of the plasma membrane and binds with high affinity to the IP3Rs, the second family of proteins central to voltage-independent calcium homeostasis. The ~300kDa IP3Rs are membrane proteins in‐ serted into the endoplasmic reticulum of non-excitable cells and the SR of excitable cells, and are active as tetramers. IP3Rs are calcium release channels that regulate the egress of pools of calcium stored within the lumen of the endoplasmic reticulum or the SR. The IP3R calcium release process is highly regulated and depends on factors including lumen calcium content, cytosolic free calcium, the post-translational modification of the receptor, and the binding of regulator proteins like bcl-2 [71]. IP3R calcium release contributes to cytosolic cal‐ cium signaling events through the information encoded in the amplitude of released calci‐

um entering cells through the TRP channels spark downstream signaling responses to receptor stimulation or to environmental challenges. As muscle stretch and oxidant stress contribute to the pathophysiology of atrial fibrillation [77], calcium entry linked to TRP channels may contribute to the hypertrophy and fibrosis that accompany atrial fibrillation.

Voltage-Independent Calcium Channels, Molecular Sources of Supraventricular Arrhythmia

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

95

In 1986 Putney raised a critically important question about voltage-independent calcium ho‐ meostasis [78]. He noted that calcium release events initiated by inositol-1,4,5-trisphosphate could deplete intracellular calcium stores. This depletion would disrupt continued calcium signaling. Putney proposed that cells must contain a mechanism to sense the calcium con‐ tent of their stores and promote calcium entry in response to store depletion. This logical proposition was widely accepted. Electrophysiological and calcium imaging protocols clear‐ ly demonstrate that depleting cell calcium stores in calcium-free media provokes a dramatic calcium entry when external calcium is restored to these cells. That is, calcium store deple‐ tion activates a cell mechanism to replenish these stores. The initial hypotheses to explain SOCC calcium entry included a calcium-inducible factor, a direct-coupling mechanism be‐ tween the store and the channel involving the actin cytoskeleton, and an indirect coupling mechanism [79]. In 2005, however, the elegant molecular mechanism for SOCC calcium en‐ try came into focus. Dziadek and colleagues [80] identified stromal interaction molecule 1 (Stim1) which subsequently was shown to be a sensor for the lumenal calcium of the endo‐ plasmic reticulum. Stim1 resides mainly in the endoplasmic reticulum, contains a single transmembrane domain, and has a calcium-binding EF-hand domain positioned within the lumen of the endoplasmic reticulum. In unstimulated cells, Stim1 distributes throughout the endoplasmic reticulum membrane. Depletion of calcium from the endoplasmic reticulum lu‐ men by any mechanism including calcium release through the IP3R causes Stim1 to translo‐ cate to plasma membrane-endoplasmic reticulum junctions. Here Stim1 docks with plasma membrane SOCCs and activates SOCC calcium entry which repletes cell calcium stores [81].

Controversy exists about the exact molecular constituents of the SOCC. In one current para‐ digm plasma membrane Orai1 proteins constitute the SOCC. This model proposes that Or‐ ai1 distributes throughout the plasma membrane of cells with full calcium stores, calcium replete cells, and is inactive. Calcium store depletion causes Stim1 to translocate to endo‐ plasmic reticulum-plasma membrane junctions. Stim1 there binds and activates Orai1 to al‐ low calcium entry. In this model the active channel is an Orai1 tetramer [82]. An alternate model posits a complex of Orai1 and TRPC1 or other isoforms of TRPC as the active calcium channel which responds to cell store-depletion but not to extracellular depolarizing influen‐ ces [75]. Regardless of this debate, the Orai proteins are the fourth family of proteins critical to voltage-independent calcium homeostasis whose tightly regulated function allows contin‐

How might this general SOCC pathway relate to current views of arrhythmia? As one exam‐ ple, SR calcium store depletion through 'leaky' ryanodine receptor might initiate [Stim1-Or‐ ai1/TRPC1] voltage-independent calcium entry in an effort to maintain SR or other myocyte calcium stores. This type of calcium entry may exacerbate the driving force for afterdepolari‐ zation posited by Marks and others(Figure 5). It also might provoke more serious unexpect‐

ued physiological calcium signaling.

ed forms of electrical instability which we outline below.

**Figure 4**: **Model of Gαq Signaling**. Agonist occupation of a specific Gαq receptor proteins activate a phosphatidylinositol specific phospholipase C (PLC-PI). Active lipase hydrolyzes plasma membrane phosphatidylinositol. This produces diacylglycerol and inositol-1,4,5-trisphosphate. Both intermediates activate calcium signaling; the former via plasma membrane TRPC3, the latter by **Figure 4. Model of Gag Signaling.** Agonist occupation of specific Gαq receptor proteins activate a phosphatidylinosi‐ tol specific phospholipase C (PLC-Pl). Active lipase hydrolyzes plasma membrane phosphatidylinositol. This produces diacylglycerol and inositol-1,4,5-trisphosphate. Both intermediates activate calcium signaling: the former via plasma membrane TRPC3, the latter by binding to the IP3R which initiates SR/ER calcium release.

um and the frequency at which release occurs. Whether the IP3Rs and the ryanodine receptor access identical calcium stores in excitable cells remains an actively investigated question.

binding to the IP3R which initiates SR/ER calcium release.

Diacylglycerol the second signaling intermediate is hydrophobic so it remains intercalated in membranes following its release from plasma membrane phosphatidylinositol. Diacylgly‐ cerol first was believed to signal by activating a protein kinase C. Subsequent work has shown that it also activates members of the third family of voltage-independent calcium sig‐ naling proteins which may be germane to arrhythmia, the TRPC family of calcium channels [72,73].

The TRP channels were first identified in drosophila where they play a central role in vision transduction [74]. Subsequent work from the laboratories of Birnbaumer [75], Montell [76], and others identified multiple families of mammalian TRP channels including the classical (TRPC), the melatonin, the vallinoid, and the ankyrin repeat forms. TRP channels contain six transmembrane domains and an ion pore domain which selects calcium over sodium under most conditions. Diacylglycerol released following Gαq receptor stimulation binds to TRPC3 and TRPC6, activates these channels, and permits cell calcium entry. The TRPC1 channel may participate in cell signaling as a subunit of the store-operated calcium channel (SOCC) which maintains cell calcium stores [75]. The TRPM3 &TRPA channels appear to ac‐ tivate when cells are stretched while TRPM2 responds to increased oxidant stress [75]. Calci‐ um entering cells through the TRP channels spark downstream signaling responses to receptor stimulation or to environmental challenges. As muscle stretch and oxidant stress contribute to the pathophysiology of atrial fibrillation [77], calcium entry linked to TRP channels may contribute to the hypertrophy and fibrosis that accompany atrial fibrillation.

In 1986 Putney raised a critically important question about voltage-independent calcium ho‐ meostasis [78]. He noted that calcium release events initiated by inositol-1,4,5-trisphosphate could deplete intracellular calcium stores. This depletion would disrupt continued calcium signaling. Putney proposed that cells must contain a mechanism to sense the calcium con‐ tent of their stores and promote calcium entry in response to store depletion. This logical proposition was widely accepted. Electrophysiological and calcium imaging protocols clear‐ ly demonstrate that depleting cell calcium stores in calcium-free media provokes a dramatic calcium entry when external calcium is restored to these cells. That is, calcium store deple‐ tion activates a cell mechanism to replenish these stores. The initial hypotheses to explain SOCC calcium entry included a calcium-inducible factor, a direct-coupling mechanism be‐ tween the store and the channel involving the actin cytoskeleton, and an indirect coupling mechanism [79]. In 2005, however, the elegant molecular mechanism for SOCC calcium en‐ try came into focus. Dziadek and colleagues [80] identified stromal interaction molecule 1 (Stim1) which subsequently was shown to be a sensor for the lumenal calcium of the endo‐ plasmic reticulum. Stim1 resides mainly in the endoplasmic reticulum, contains a single transmembrane domain, and has a calcium-binding EF-hand domain positioned within the lumen of the endoplasmic reticulum. In unstimulated cells, Stim1 distributes throughout the endoplasmic reticulum membrane. Depletion of calcium from the endoplasmic reticulum lu‐ men by any mechanism including calcium release through the IP3R causes Stim1 to translo‐ cate to plasma membrane-endoplasmic reticulum junctions. Here Stim1 docks with plasma membrane SOCCs and activates SOCC calcium entry which repletes cell calcium stores [81].

Controversy exists about the exact molecular constituents of the SOCC. In one current para‐ digm plasma membrane Orai1 proteins constitute the SOCC. This model proposes that Or‐ ai1 distributes throughout the plasma membrane of cells with full calcium stores, calcium replete cells, and is inactive. Calcium store depletion causes Stim1 to translocate to endo‐ plasmic reticulum-plasma membrane junctions. Stim1 there binds and activates Orai1 to al‐ low calcium entry. In this model the active channel is an Orai1 tetramer [82]. An alternate model posits a complex of Orai1 and TRPC1 or other isoforms of TRPC as the active calcium channel which responds to cell store-depletion but not to extracellular depolarizing influen‐ ces [75]. Regardless of this debate, the Orai proteins are the fourth family of proteins critical to voltage-independent calcium homeostasis whose tightly regulated function allows contin‐ ued physiological calcium signaling.

um and the frequency at which release occurs. Whether the IP3Rs and the ryanodine receptor access identical calcium stores in excitable cells remains an actively investigated

**Figure 4**: **Model of Gαq Signaling**. Agonist occupation of a specific Gαq receptor proteins activate a phosphatidylinositol specific phospholipase C (PLC-PI). Active lipase hydrolyzes plasma membrane phosphatidylinositol. This produces diacylglycerol and inositol-1,4,5-trisphosphate. Both intermediates activate calcium signaling; the former via plasma membrane TRPC3, the latter by

**Figure 4. Model of Gag Signaling.** Agonist occupation of specific Gαq receptor proteins activate a phosphatidylinosi‐ tol specific phospholipase C (PLC-Pl). Active lipase hydrolyzes plasma membrane phosphatidylinositol. This produces diacylglycerol and inositol-1,4,5-trisphosphate. Both intermediates activate calcium signaling: the former via plasma

**Gαq**

**PLC-PI**

Ca

Ca

**SERCA**

**IP3R**

Ca

Ca

Ca

Ca

**TRPC3**

**Ca**

Ca

**Ca**

Ca

**Calcium signal**

Ca

Ca

*IP3R*

Ca

Ca

membrane TRPC3, the latter by binding to the IP3R which initiates SR/ER calcium release.

**SERCA**

binding to the IP3R which initiates SR/ER calcium release.

Ca

Ca Ca

Ca Ca

*PI TRPC3*

**Gαq** *PLC-*

94 Atrial Fibrillation - Mechanisms and Treatment

Phosphatidyl inositol

Gαq agonist: e.g. norepinephrine

Inositol-1,4,5 trisphosphate

Diacylglycerol

Diacylglycerol the second signaling intermediate is hydrophobic so it remains intercalated in membranes following its release from plasma membrane phosphatidylinositol. Diacylgly‐ cerol first was believed to signal by activating a protein kinase C. Subsequent work has shown that it also activates members of the third family of voltage-independent calcium sig‐ naling proteins which may be germane to arrhythmia, the TRPC family of calcium channels

The TRP channels were first identified in drosophila where they play a central role in vision transduction [74]. Subsequent work from the laboratories of Birnbaumer [75], Montell [76], and others identified multiple families of mammalian TRP channels including the classical (TRPC), the melatonin, the vallinoid, and the ankyrin repeat forms. TRP channels contain six transmembrane domains and an ion pore domain which selects calcium over sodium under most conditions. Diacylglycerol released following Gαq receptor stimulation binds to TRPC3 and TRPC6, activates these channels, and permits cell calcium entry. The TRPC1 channel may participate in cell signaling as a subunit of the store-operated calcium channel (SOCC) which maintains cell calcium stores [75]. The TRPM3 &TRPA channels appear to ac‐ tivate when cells are stretched while TRPM2 responds to increased oxidant stress [75]. Calci‐

question.

[72,73].

How might this general SOCC pathway relate to current views of arrhythmia? As one exam‐ ple, SR calcium store depletion through 'leaky' ryanodine receptor might initiate [Stim1-Or‐ ai1/TRPC1] voltage-independent calcium entry in an effort to maintain SR or other myocyte calcium stores. This type of calcium entry may exacerbate the driving force for afterdepolari‐ zation posited by Marks and others(Figure 5). It also might provoke more serious unexpect‐ ed forms of electrical instability which we outline below.

second, the arachidonate this lipase produces may activate ARC, voltage-independent calci‐

Voltage-Independent Calcium Channels, Molecular Sources of Supraventricular Arrhythmia

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

97

The past 50 years of research in heart calcium and arrhythmogenesis have focused principal‐ ly on voltage-dependent calcium homeostasis. Indeed there are only few reports which identify atrial, ventricular, sinoatrial or Purkinje cell expression of the molecular constitu‐ ents of voltage-independent calcium homeostasis. Even fewer of these reports detail the unique intracellular distribution of these proteins in the different types of heart cells or

Bootman and co-authors [84] provide convincing evidence that atrial myocytes contain pre‐ dominately the type 2 IP3R. They show that atrial myocytes express about 10-fold more IP3R than do ventricular myocytes. An impressive observation they and others report is that this calcium release channel distributes mainly in the junctional SR near to the sarcolemmal membrane and that these IP3Rs associate with the junctional ryanodine receptors that encir‐ cle each atrial myocyte. Bootman and others suggest that these IP3Rs sensitize ryanodine re‐ ceptor calcium release and may participate in the response of atria to inotropic Gαq receptor

Only little is known about the expression of the TRP channels, the Orai channels, and the Stim proteins in normal atrial muscle and pulmonary veins. To our knowledge how patho‐ logical situations like paroxysmal or sustained atrial fibrillation affect the expression of these calcium channels and channel regulators has not been investigated. This is important infor‐ mation as these families of proteins control the induction of hypertrophy, the response to stretch, fibrosis, and the intrinsic pathway for apoptosis. A complete evaluation of these sig‐ naling proteins in normal and diseased atria would dissect the molecular mechanisms through which atria responds to clinically relevant stressors and how these responses may

Ventricular myocytes contain much lower levels of the IP3Rs relative to atria. Of interest Mohler [85] and others report that ventricular IP3Rs preferentially associate with the para‐ junctional SR of the T-tubule. The purpose or consequence of the specific localization of these calcium release channels is actively investigated. The responsiveness of heart muscle to Gαq stimulation increases during hypertrophy and heart failure. These results are in keeping with reports that the expression of ventricular IP3Rs increases in these diseases. Us‐ ing probes specific for the type 1 IP3R, Marks [86] showed that the ventricular content of these channels nearly triples in failing heart while characteristically the content of the ryano‐ dine receptor decreases by a factor of at least two. Little is known about the expression or functional properties of ventricular TRP channels, Orai channels, and Stim proteins either in

By comparison with the paucity of work in ventricular myocytes, in 1994 Volpe [87] provid‐ ed the first evidence that IP3Rs are highly expressed in the conduction system. Subsequent elegant and thorough analyses by Boyden, ter Keurs and colleagues demonstrated an intri‐ cate distribution of the IP3Rs and the ryanodine receptors in the Purkinje cells of the con‐ duction system [88]. Much like atrial myocytes, the IP3Rs distribute at the periphery of

um entry, the production of inflammatory molecules, and possibly ectopic activity.

study how this pattern of distribution might contribute to arrhythmogenesis.

favor dysfunction including electrical instability like atrial fibrillation.

agonists.

normal or diseased heart.

**Figure 5. Potential Interaction between Orai Calcium Entry and Arrhythmogenic SR Calcium Leak.** Calcium leak through the ryanodine receptor. (*RyR & right green arrow*) depletes SR calcium. This depletion may activate Oraillinked calcium entry (*Left green arrow*) to maintain E-C patency & muscle function. A futile cycle of calcium entry-leak may exacerbate NCX-linked calcium efflux and cell depolarization, driving it more frequently or more quickly toward threshold (*Rightmost black arrows*).

Orai2 and Orai3 are the remaining members of this fourth family. Orai2 is a pseudogene and has garnered some interest. By contrast, Shuttleworth first demonstrated that Orai3 is an im‐ portant participant in voltage-independent calcium signaling [64]. Elegant work from his lab group shows that pentamers of Orai3 and Orai1 form an arachidonate regulated calcium channel (ARC). Arachidonate binding to ARC causes channel activation and permits volt‐ age-independent calcium entry. Like the SOCC, ARC also requires Stim1 but it uses the small pool of Stim1 present in the plasma membrane. The arachidonate which activates ARC can arise from several sources. In cell culture experiments it is usually added exoge‐ nously. Calcium-dependent cytosolic phospholipase A2 is a key source of cellular free arach‐ idonate in physiological settings. Importantly, the arachidonate arising from the action of calcium-dependent cytosolic phospholipase A2 on cell phospholipids is a key source for in‐ flammatory prostaglandins and leukotrienes. CaMKII phosphorylates and activates this phospholipase [83]. Thus two possibilities emerge. First, myocyte calcium loading may acti‐ vate CaMKII which then phosphorylates cytosolic calcium-dependent phospholipase A2; second, the arachidonate this lipase produces may activate ARC, voltage-independent calci‐ um entry, the production of inflammatory molecules, and possibly ectopic activity.

The past 50 years of research in heart calcium and arrhythmogenesis have focused principal‐ ly on voltage-dependent calcium homeostasis. Indeed there are only few reports which identify atrial, ventricular, sinoatrial or Purkinje cell expression of the molecular constitu‐ ents of voltage-independent calcium homeostasis. Even fewer of these reports detail the unique intracellular distribution of these proteins in the different types of heart cells or study how this pattern of distribution might contribute to arrhythmogenesis.

Bootman and co-authors [84] provide convincing evidence that atrial myocytes contain pre‐ dominately the type 2 IP3R. They show that atrial myocytes express about 10-fold more IP3R than do ventricular myocytes. An impressive observation they and others report is that this calcium release channel distributes mainly in the junctional SR near to the sarcolemmal membrane and that these IP3Rs associate with the junctional ryanodine receptors that encir‐ cle each atrial myocyte. Bootman and others suggest that these IP3Rs sensitize ryanodine re‐ ceptor calcium release and may participate in the response of atria to inotropic Gαq receptor agonists.

Only little is known about the expression of the TRP channels, the Orai channels, and the Stim proteins in normal atrial muscle and pulmonary veins. To our knowledge how patho‐ logical situations like paroxysmal or sustained atrial fibrillation affect the expression of these calcium channels and channel regulators has not been investigated. This is important infor‐ mation as these families of proteins control the induction of hypertrophy, the response to stretch, fibrosis, and the intrinsic pathway for apoptosis. A complete evaluation of these sig‐ naling proteins in normal and diseased atria would dissect the molecular mechanisms through which atria responds to clinically relevant stressors and how these responses may favor dysfunction including electrical instability like atrial fibrillation.

Ventricular myocytes contain much lower levels of the IP3Rs relative to atria. Of interest Mohler [85] and others report that ventricular IP3Rs preferentially associate with the para‐ junctional SR of the T-tubule. The purpose or consequence of the specific localization of these calcium release channels is actively investigated. The responsiveness of heart muscle to Gαq stimulation increases during hypertrophy and heart failure. These results are in keeping with reports that the expression of ventricular IP3Rs increases in these diseases. Us‐ ing probes specific for the type 1 IP3R, Marks [86] showed that the ventricular content of these channels nearly triples in failing heart while characteristically the content of the ryano‐ dine receptor decreases by a factor of at least two. Little is known about the expression or functional properties of ventricular TRP channels, Orai channels, and Stim proteins either in normal or diseased heart.

Orai2 and Orai3 are the remaining members of this fourth family. Orai2 is a pseudogene and has garnered some interest. By contrast, Shuttleworth first demonstrated that Orai3 is an im‐ portant participant in voltage-independent calcium signaling [64]. Elegant work from his lab group shows that pentamers of Orai3 and Orai1 form an arachidonate regulated calcium channel (ARC). Arachidonate binding to ARC causes channel activation and permits volt‐ age-independent calcium entry. Like the SOCC, ARC also requires Stim1 but it uses the small pool of Stim1 present in the plasma membrane. The arachidonate which activates ARC can arise from several sources. In cell culture experiments it is usually added exoge‐ nously. Calcium-dependent cytosolic phospholipase A2 is a key source of cellular free arach‐ idonate in physiological settings. Importantly, the arachidonate arising from the action of calcium-dependent cytosolic phospholipase A2 on cell phospholipids is a key source for in‐ flammatory prostaglandins and leukotrienes. CaMKII phosphorylates and activates this phospholipase [83]. Thus two possibilities emerge. First, myocyte calcium loading may acti‐ vate CaMKII which then phosphorylates cytosolic calcium-dependent phospholipase A2;

**Figure 5. Potential Interaction between Orai Calcium Entry and Arrhythmogenic SR Calcium Leak.** Calcium leak through the ryanodine receptor. (*RyR & right green arrow*) depletes SR calcium. This depletion may activate Oraillinked calcium entry (*Left green arrow*) to maintain E-C patency & muscle function. A futile cycle of calcium entry-leak may exacerbate NCX-linked calcium efflux and cell depolarization, driving it more frequently or more quickly toward

RyR

**SCC**

**Orai**

Ca

Ca

96 Atrial Fibrillation - Mechanisms and Treatment

**SERCA**

Ca

Ca

Ca

Ca

**SERCA**

Ca

threshold (*Rightmost black arrows*).

3Na

Ca

Ca Ca 3Na **+**

**++**

Leaky ryanodine receptor ( ) SR store depletion Orai1 activation 'Futile cycle' of calcium leak-load Exacerbate NCX inward current Favor arrhythmogenesis

**NCX**

Ca

Ca

Ca

By comparison with the paucity of work in ventricular myocytes, in 1994 Volpe [87] provid‐ ed the first evidence that IP3Rs are highly expressed in the conduction system. Subsequent elegant and thorough analyses by Boyden, ter Keurs and colleagues demonstrated an intri‐ cate distribution of the IP3Rs and the ryanodine receptors in the Purkinje cells of the con‐ duction system [88]. Much like atrial myocytes, the IP3Rs distribute at the periphery of 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].

Together these data support and extend earlier intact animal studies and provide striking evidence that voltage-independent calcium homeostasis contributes to atrial arrhythmogen‐

Voltage-Independent Calcium Channels, Molecular Sources of Supraventricular Arrhythmia

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

99

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

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

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‐

ic calcium signaling.

signaling events are involved.

of wavebreak to account for this unexpected result.

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‐ mal atrial fibrillation.
