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

**Transient receptor potential proteins**

> **TRP C3**

**Gαq signaling Autonomic overload Hypertrophy**

Extra‐ and intra‐cellular inputs Induce Ca++ entry

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

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

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

Reports in the literature and our data suggest they do. Myocytes and Purkinje cells express the cellular calcium transporters, kinases, lipases and other proteins that initiate and regu‐ late voltage-independent calcium entry and calcium signaling. These include the IP3Rs, the

**Stretch Redox**

**TRP M2**

**Arachidonate‐regulated calcium channel: (Orai1/Orai3 pentamer)**

**Orai1 Orai3**

**Arachidonate**

Increase cytosolic Ca++ Activate phospholipase A2 Release arachidonate Activate ARC ARC Ca++ entry

**stress**

**TRP A**

**Orai1**

**ER/SR**

**RyR**

**Store‐operated calcium channel (Orai1 tetramer)**

90 Atrial Fibrillation - Mechanisms and Treatment

**IP3R**

ER/SR Ca++ depletes Activates the SOCC Refills Ca++ stores

noid metabolism (*Right box*).

signals (Figure 3).

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

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

activate this cryptic arrhythmogenic signaling pathway. The novel fourth mechanism out‐

Voltage-Independent Calcium Channels, Molecular Sources of Supraventricular Arrhythmia

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

93

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‐

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‐

lined below satisfies the requirements for a purely focal hypothesis for arrhythmia.

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

pendent calcium channels can provoke heart muscle electrical instability.

**homeostasis**

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 produce sporadic or high frequency myocardial electrical instability.
