Purkinje Cells from RyR2 Mutant Mice are Highly Arrhythmogenic but Responsive to Targeted Therapy

Abstract

Rationale

The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca²⁺ release events in catecholaminergic polymorphic ventricular tachycardia (CPVT) yet evidence supporting this mechanism at the cellular level is lacking.

Objective

We sought to determine the frequency and severity of spontaneous Ca²⁺ release events and the response to the anti-arrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2^R4496C/+ CPVT mutant mice and littermate controls.

Methods and Results

We crossed RyR2^R4496C/+ knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated VMs (EGFP-) and Purkinje cells (EGFP+) from wild type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca²⁺ dynamics recorded by microfluorimetry. Both wild type and RyR2^R4496C/+ mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and τ_decay in the mutant Purkinje cells was significantly slower than that observed in wild type Purkinje cells. Of the four groups studied, RyR2^R4496C/+ mutant Purkinje cells were also most likely to develop spontaneous Ca²⁺ release events and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, while all four groups showed increases in the frequency of arrhythmogenic Ca²⁺_i events, the RyR2^R4496C/+ Purkinje cells responded with the most profound abnormalities in intracellular Ca²⁺ handling, including a significant increase in the frequency of unstimulated Ca²⁺_i events, the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild type mice showed suppression of spontaneous Ca²⁺ release events with flecainide, whereas in RyR2^R4496C/+ mice, the Purkinje cells were preferentially responsive to drug. In contrast, the RyR2 blocker tetracaine was equally efficacious in mutant Purkinje cells and ventricular myocytes.

Conclusions

Purkinje cells display a greater propensity to develop abnormalities in intracellular Ca²⁺ handling than ventricular myocytes. This pro-arrhythmic behavior is enhanced by disease-causing mutations in the RyR2 Ca²⁺ release channel and greatly exacerbated by catecholaminergic stimulation, with the development of arrhythmogenic triggered beats. These data support the concept that Purkinje cells are critical contributors to arrhythmic triggers in animal models and humans with CPVT and suggest a broader role for the Purkinje fiber network in the genesis of ventricular arrhythmias.

Introduction

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited syndrome characterized by adrenergically mediated ventricular tachycardias, often resulting in syncope and sudden cardiac death¹. First described more than 20 years ago by Coumel, recent genetic studies have demonstrated that mutations in the ryanodine receptor (RyR2) and calsequestrin (CASQ2) genes underlie some cases of this syndrome^{2, 3}, implicating aberrant control of Ca²⁺ homeostasis in the molecular pathogenesis.

The specialized cardiac conduction system (CCS) comprises a heterogeneous network of cells that orchestrate the initiation and propagation of a wave of electrical excitation throughout the myocardium^{4, 5}. Purkinje cells (PCs) are the most distal component of the CCS and they deliver the depolarizing impulse to the working myocytes of the ventricular myocardium. More than forty years ago, Hoffman and colleagues proposed that PCs might “be a significant factor in human arrhythmias⁶.” Since that time, substantial, but somewhat indirect experimental data has accumulated supporting the concept that PCs play a key mechanistic role triggering a broad range of life-threatening ventricular arrhythmias. Indeed, recent optical mapping data of RyR2^R4496C/+ mutant mice, which to a remarkable extent phenocopy human CPVT, support the hypothesis that Ca²⁺-dependent arrhythmic triggers may be arising from the Purkinje fiber network⁷.

A mechanistic basis for the pro-arrhythmic behavior of PCs is slowly emerging. Underlying ultrastructural features such as a decreased T-tubular density⁸, the presence of IP₃-sensitive Ca²⁺ channels⁹, and susceptibility to Ca²⁺ overload due to greater Na⁺ load¹⁰ may in part account for the propensity of these specialized cells to develop abnormal Ca²⁺ release events. Moreover, recent data suggest that higher diastolic Ca_i-voltage coupling gain in Purkinje fibers may underlie the generation delayed after-depolarizations (DADs) and triggered beats¹¹.

In addition to these cell-autonomous pro-arrhythmic properties, the macroscopic structural features of the specialized ventricular conduction system network may also contribute to the enhanced capacity of PCs to trigger arrhythmias. While electrical loading will normally prevent spontaneous Ca²⁺ release events (SCaEs) in ventricular myocytes (VMs) from evoking propagated action potentials, the same is not true of events arising within PCs. Electronic interactions between PCs and the underlying myocardium at Purkinje-ventricular junctions (PVJ) are limited, such that stochastic Ca²⁺ release events in PCs may trigger APs that successfully propagate into working myocytes^12-14. Moreover, propagation across the PV junction is highly sensitive to the extent of coupling, as evidenced by a variety of experimental approaches including computational studies and genetically engineered mouse model systems^{12, 15}.

As a consequence of these multi-scale mechanisms, when challenged with stressors that promote electrical remodeling or changes in intracellular Ca²⁺ handling, such as mutations in Ca²⁺-handling proteins as seen in CPVT, PCs may be preferentially “primed” to develop arrhythmic triggers^16-22.

Through a transcriptional screen our laboratory recently identified a novel cell adhesion molecule named Cntn2, which is specifically expressed in cells of the specialized cardiac conduction system, including the distal Purkinje fiber network²³. Moreover, we demonstrated that genetically engineered mice harboring a Cntn2-EGFP BAC transgene faithfully recapitulated expression of the endogenous protein, thereby providing a tool to specifically identify and distinguish PCs from working SCaEs. Interestingly, in our initial study we found that even wild type PCs were more arrhythmogenic than VMs, with more frequent unstimulated Ca²⁺_i release events, DADs and triggered beats. Using this same approach, we now have examined intracellular Ca²⁺ dynamics in dissociated adult PCs and VMs from both wild type and RyR2^R4496C/+ mutant mice, at baseline and following catecholaminergic stimulation as might be seen during arrhythmic episodes in CPVT. We also determined the anti-arrhythmic effects of RyR2 blockers in PCs and VMs from wild type and mutant mice.

Methods

An expanded Methods section is available in the Online Data Supplement.

Mutant Mice

All experiments were performed according to protocols approved by the NYU School of Medicine Institutional Animal Care and Use Committee and conformed to the National Institutes of Health (NIH) guidelines for the care and use of Laboratory Animals. Cntn2-EGFP BAC transgenic mice and RyR2^R4496C/+ mutant mice have both been previously described^{23, 24}. All mice studied were F1 crosses between the two strains and used at 8-12 weeks of age. To minimize experimental variability, data from VMs and PCs from the same preparation were always collected on the same day.

Immunohistochemistry

Dissociated myocytes from adult hearts were stained as previously described²³. Primary antibodies were directed again ankyrin-G, Scn5a and α-actinin and visualized by confocal microscopy.

Ca²⁺ imaging and voltage measurements

Adult myocytes were isolated using an established enzymatic digestion protocol²⁵ and intracellular Ca²⁺ imaging was performed using a microfluorimetry system (IonOptix, Corp), as previously described²³. Spontaneous Ca²⁺ release events (SCaEs) were defined as unstimulated increases in intracellular Ca²⁺ and were quantified during the 6 second unpaced interval following the 10 beat pacing train. Transmembrane potential were recorded in whole cell current-clamp mode (Axon Instruments), also as previously described^23,25.

Statistics

Kinetic parameters were compared using Student's t-test. The proportion of cells displaying SCaEs was compared using Fisher's exact test. The number of SCaEs per cell was compared by the Mann-Whitney test. P values < 0.05 were considered significant.

Results

Identification of Adult Murine Purkinje Cells

We previously demonstrated that the Cntn2-EGFP BAC transgenic mouse strain could be used to specifically identify the relatively rare PCs from a mixed population of dissociated ventricular cells²³. The PCs could be visualized both in situ, in the subendocardial region of the ventricles (Figure 1A) and in dissociated cells, where both VMs and PCs express the cardiac specific proteins Scn5a and sarcomeric α-actinin (Figure 1B-E), but only the PCs express EGFP.

Figure 1. Visualization of Murine Purkinje Cells.

Immunofluorescent image (A) of adult Cntn2-EGFP transgenic mouse ventricle showing membrane-enriched staining of ankyrin-G (red) and intrinsic EGFP fluorescence (green). Typical subendocardial location of Purkinje cells is seen. Paired phase (B) and epifluorescent (C) image showing an EGFP-negative ventricular myocyte (VM) and EGFP-positive Purkinje cell (PC) in the same field. Ventricular myocyte (D) and Purkinje cell (E) co-stained for Scn5a (red) and α-actinin (blue). Individual channels are shown below. Both cell types express the two cardiac markers, but EGFP fluorescence is only observed in the Purkinje cell. Magnification is indicated.

Kinetic Properties in Purkinje Cells and Ventricular Myocytes

Fluorescence photometry with the calcium-sensitive dye x-Rhod-1 was used to study intracellular Ca²⁺ dynamics (Figure 2). Confirming our previous observation²³, wild type PCs displayed significantly slower kinetics of activation and relaxation compared to VMs, with rise times of 117 ± 6.6 ms (n=8) vs. 68 ± 2.3 (n=10); p<0.00001; and τ_decay of 225 ± 24 ms (n=8) vs. 155 ± 10 (n=10); p<0.01. A similar large difference in kinetics was observed comparing the RyR^R4496C/+ mutant PCs and mutant myocytes, with rise times of 129 ± 6.5 ms (n=10) vs. 71 ± 5.4 ms (n=10); p<0.000002; and τ_decay of 312 ± 23 ms (n=10) vs. 201 ± 15 ms (n=10); p<0.001. Interestingly, the τ_decay in the mutant PCs was significantly longer than in wild type PCs (p<.01).

Figure 2. Global Intracellular Ca²⁺ Transient Kinetics.

Time to peak of the Ca²⁺ transients (A) and τ_decay (B) in wild type (WT) and RyR^R4496C/+ mutant (MUT) ventricular myocytes (VM) and Purkinje cells (PC). Examples of Ca²⁺ transients in each of the four cell types are also shown (C-F). The slower kinetics in WT and MUT PCs are evident. * p<0.05 compared to VMs of same genotype; **p<0.05 compared to WT VMs; ***p<0.05 vs. all other groups.

Spontaneous Ca²⁺ release events in RyR^R4496C/+ Purkinje Cells

We next determined the propensity of VMs and PCs to develop SCaEs following field stimulation at a range of pacing frequencies. We first examined wild type VMs and PCs. As expected, wild type VMs were quite stable; none of 15 cells studies developed SCaEs at 1 Hz and only one cell displayed unpaced SCaEs after pacing at 3Hz or 5 Hz. Wild type PCs had a greater tendency to develop unpaced SCaEs compared to the VMs, especially following higher pacing frequencies (3 of 10 cells at 3 Hz and 5 Hz), as shown in Figure 3. We next examined the behavior of cells from RyR^R4496C/+ mutant mice. Mutant VMs and mutant PCs were more arrhythmogenic than their wild type counterparts, both with respect to the proportion of cells displaying SCaEs, as well as the number of SCaEs per cell. This was especially the case for the RyR^R4496C/+ mutant PCs, with as many as 62% of cells studied showing arrhythmic activity, as shown in Figure 3 and summarized in Table 1.

Figure 3. Rate Dependence of Arrhythmogenic Spontaneous Calcium Release Events.

Proportion of cells displaying SCaEs (A, B) and number of SCaEs per cell (C, D) in dissociated VMs and PCs from WT and RyR2^R4496C/+ MUT mice. *p<0.05 for PCs compared to VMs of same genotype and pacing frequency.

Table 1. Spontaneous Calcium Release Events in Ventricular Myocytes and Purkinje Cells.

	Cells with SCaEs				SCaEs Per Cell
	- ISO			+ISO	-ISO			+ ISO
	1 Hz	3 Hz	5 Hz	1 Hz	1 HZ	3 Hz	5 Hz	1 Hz
WT VM	0% (15)	7% (15)	7% (15)	14% (7)	0 ± 0 (15)	0.07 ± .07 (15)	0.07 ± .07 (15)	0.14 ± .14 (7)
WT PC	10% (10)	30% (10)	30% (10)	57% (7)	0.10 ± .10 (10)	0.40 ± .22 (10)	0.40 ± .22 (10)	3.00 ± 1.38 (7)
MUT VM	22% (41)	24% (41)	29% (41)	40% (10)	0.29 ± .09 (41)	0.37 ± .11 (41)	.34 ± .09 (41)	1.10 ± .48 (10)
MUT PC	52% (29)	62% (29)	62% (29)	90% (10)	1.07 ± .32 (29)	1.72 ± .36 (29)	1.65 ± .36 (29)	12.2 ± 1.48 (10)

Data are shown as percentage of of cells with spontaneous Ca²⁺ release events (SCaEs) and the mean ± SEM SCaEs/cell at each pacing frequency. The number of cells studied for each condition are shown in parentheses. WT, wild type; MUT, mutant; VM, ventricular myocyte; PC, Purkinje cell; ISO, isoproterenol.

Increased Sensitivity to Catecholaminergic Stimulation

Inasmuch as arrhythmic behavior in patients and animal models of CPVT is provoked during catecholaminergic stimulation, the effects of isoproterenol treatment (30nM) on intracellular Ca²⁺ dynamics in wild type and mutant VMs and PCs was assessed. When paced at 1 Hz, wild type VMs, wild type PCs and mutant VMs all showed modest increases in the proportion of cells that developed ScaEs compared to untreated controls, as shown in Figure 4 and Table 1. However, the effect was most profound in mutant PCs; almost all (90%; 9 of 10) developed arrhythmogenic Ca²⁺ activity. Moreover, the quantity of SCaEs per cell was significantly greater for the RyR2^R4496C PCs (12.2 ± 1.5/cell) compared to RyR2^R4496C VMs (1.1 ± .48/cell). Furthermore, even at the slow pacing frequency of 1 Hz, the PCs developed marked abnormalities in intracellular Ca²⁺ handling, including oscillations, alternans and sustained salvos of presumed triggered beats following the end of the pacing train (Figure 4 D-G). To confirm that these abnormalities in calcium regulation were indeed sufficient to trigger action potentials, we performed simultaneous Ca²⁺ imaging and recordings of transmembrane potentials in whole cell current-clamp mode. These studies indeed demonstrated that SCaEs could successfully trigger action potentials in the mutant PCs (Figure 4H). Thus, the RyR^R4496C/+ mutant PCs appeared to be especially sensitive to the pro-arrhythmic effects of catecholaminergic stimulation.

Figure 4. Effects of Isoproterenol.

Proportion of cells displaying SCaEs in VMs and PCs from WT and MUT mice after exposure to isoproterenol (A). Number of SCaEs per cell (B). Intracellular Ca²⁺ recordings showing SCaEs in a RyR2^R4496C/+ mutant VM (C) and in several RyR2^R4496C/+ mutant PCs (D-G). Simultaneous recording of [Ca²⁺]_i and transmembrane voltage demonstrate that SCaEs can lead to triggered action potentials, highlighted in boxed area (H). Arrows indicate field stimulation, which is at 1 Hz in these examples. *p< 0.05 compared to VM of same genotype.

Preferential Suppression of Spontaneous Ca²⁺ Release Events in Mutant Purkinje Cells

The anti-arrhythmic agent flecainide has shown efficacy in preventing ventricular arrhythmias in mice and humans with Casq2-associated CPVT²⁶. We therefore tested the effects of flecainide on the magnitude of spontaneous Ca²⁺ release events in VMs and PCs. In agreement with previous studies²⁷, the modest arrhythmic activity observed in wild type VMs was diminished with flecainide, both with respect to the number of cells developing SCaEs, as well as the number of SCaEs per cell. We also observed efficacy in wild type PCs from these same hearts, as shown in Figure 5 and Table 2. As expected, cells from RyR2^R4496C/+ mutant mice were substantially more arrhythmic at baseline than their wild type counterparts. Surprisingly, however, the mutant VMs appeared resistant to the anti-arrhythmic effects of flecainide, whereas the mutant PCs showed a significant reduction in the number of cells with SCaEs as well as a reduction in SCaEs per cell, as shown in Figure 5B, D. We also tested the effects of tetracaine, which preferentially reduces RyR2 open probability²⁸. In contrast to flecainide, this agent suppressed SCaEs in mutant PCs and VMs with equal efficacy, as shown in Figure 5E, F. These data are summarized in Table 2.

Figure 5. Effects of Flecainide and Tetracaine on SCaEs.

Proportion of cells displaying SCaEs (A, B, E) and number of SCaEs per cell (C, D, F) in dissociated VMs and PCs from WT and RyR2^R4496C/+ MUT mice in the absence (CON) or presence of flecainide (FLEC) or tetracaine (TETR). *p< 0.05 compared to control cells.

Table 2.

Anti-arrhythmic Effects of Flecainide and Tetracaine in Ventricular Myocytes and Purkinje Cells

	Cells with SCaEs		SCaEs Per Cell
	CON	FLEC	CON	FLEC
WT VM	14% (28)	3% (36)	.18 ± .09 (28)	.03 ± .03 (36)
WT PC	45% (20)	11% (18)	.80 ± .24 (20)	.22 ± .17 (18)
MUT VM	43% (56)	34% (71)	.68 ± .13 (56)	.61 ± .12 (71)
MUT PC	76% (25)	27% (37)	2.64 ± .47 (25)	.57 ± .19 (37)
	CON	TETR	CON	TETR
MUT VM	54% (24)	16% (25)	.79 (24)	.16 (25)
MUT PC	89% (19)	27% (11)	3.58 (19)	.73 (11)

Data are shown as percentage of of cells with spontaneous Ca²⁺ release events (SCaEs) and the mean ± SEM SCaEs/cell. The number of cells studied for each condition are shown in parentheses.WT, wild type; MUT, mutant; VM, ventricular myocyte; PC, Purkinje cell.

Discussion

Recent data suggest that SR Ca²⁺-release channels comprised of the CPVT-causing RyR2^R4496C mutant protein display increased sensitivity to both cytosolic and luminal Ca²⁺, predisposing them to the development of arrhythmogenic DADs and triggered beats^29-32. In this study we show that PCs from mice harboring this same mutation are preferentially and exquisitely sensitive to this underlying molecular defect. Compared to genetically identical VMs, the mutant PCs show more profound abnormalities in intracellular Ca²⁺ handling, including the development of spontaneous Ca²⁺ release events at baseline and, as demonstrated by simultaneous calcium and voltage measurements, the progression to triggered beats in response to relatively modest catecholaminergic stimulation.

This study extends our prior observation indicating that even wild type PCs are more likely to develop DADs and triggered beats compared to VMs²³. The basis for this heightened proclivity of PCs to develop arrhythmic behavior is not fully known, although the diminished T-tubular density⁸, altered action potential morphology³³, altered kinetics of Ca²⁺ handling (Fig 2) and increased intracellular Na⁺ load¹⁰ may all be contributory factors. In addition, the participation of IP₃-sensitive Ca²⁺ channels as part of the excitation-contraction coupling machinery may also influence this arrhythmogenic behavior⁹. As such, when confronted with another perturbation, in this case a defective intracellular Ca²⁺ release channel, spontaneous Ca²⁺ release events may arise. If of sufficient number, magnitude or location, these events may result in DADs. Moreover, PCs appear especially vulnerable to the development of DADs by virtue of their increased diastolic Ca_i-voltage coupling gain, behavior which may be due in part to diminished inwardly rectifying potassium currents (I_K1)¹¹. Furthermore, in contrast to well-coupled and electrically loaded working myocytes, in PCs, oscillations in membrane potential are uniquely capable of evoking propagated action potentials that can traverse the Purkinje-ventricular junction into the myocardium proper¹².

Our analysis of myocytes from RyR2^R4496C mutant mice strongly support the recent suggestion that the Purkinje network may serve as the arrhythmogenic trigger in CPVT associated with RyR2 mutations. This initial hypothesis was based in part upon the characteristic bidirectional ventricular tachycardia seen in patients and mice with this syndrome^{24, 34} and data demonstrating that ablation of the right ventricular subendocardium (where Purkinje fibers are found) with Lugol's solution converted the biventricular tachycardia to a monomorphic arrhythmia⁷. Whether or not PCs serve as arrhythmic triggers in CPVT associated with mutations in the calsequestrin gene remains to be seen^{35, 36}, but comparable studies in Casq2 mutant mice should be informative. More broadly, the Purkinje fiber network has been implicated in the initiation of ventricular tachycardia not only in CPVT, but following myocardial infarction, in dilated cardiomyopathies, in idiopathic ventricular fibrillation, in post-shock arrhythmias, and other inherited channelopathies including Brugada and long QT syndromes^{7, 37-42}. A similar analysis of PC behavior at the cellular level of resolution in these diseases should be equally revealing.

Several experimental and clinically approved agents have recently been shown to inhibit arrhythmogenic Ca²⁺ waves^{26, 27, 43}. In particular, the class 1C anti-arrhythmic agent flecainide was shown to inhibit RyR2 channel activity by reducing the duration of channel openings, especially when the RyR2 channel was activated by high luminal Ca²⁺ concentration. Moreover, flecainide showed efficacy in preventing ventricular arrhythmias in mice and in humans with Casq2-associated CPVT²⁶. Here we show that flecainide is indeed effective in suppressing spontaneous Ca²⁺ release events not only in VMs but in PCs as well. Unexpectedly, however, whereas flecainide appears equally efficacious in VMs and PCs from wild type mice, in RyR2^R4496C/+ mutant mice the beneficial effect appears restricted to only the PCs. The suppressive effect we observed in PCs occurs at substantially lower concentrations than the reported IC₅₀ for inhibition of RyR2 open probability²⁷. Conceivably, flecainide may be acting through additional targets such as voltage-gated sodium channels, secondarily influencing intracellular Ca²⁺ load and the propensity for arrhythmogenic SCaEs. Interestingly, we have recently found that Na_v1.8/Scn10a is preferentially expressed in PCs, which could account for the differential sensitivity of these cells to flecainide compared to VMs. Indeed, with the more pure RyR2 inhibitor tetracaine, we observed equal efficacy suppressing SCaEs in both VMs and PCs. Accordingly, it will be particularly informative to see if flecainide has clinical utility not only in Casq2-associated CPVT, but also in RyR2-dependent forms of this syndrome.

This study was facilitated by our recent discovery of Cntn2 in the specialized conduction system and the recognition that Cntn2-EGFP mice could facilitate the identification and isolation of conduction system cells, including PCs²³. This approach is similar in principal to using Cx40-EGFP knock-in mice⁴⁴, although that strategy may be less specific, as Cx40 is expressed not only in the conduction system, but also in atrial myocytes, and vascular endothelium and smooth muscle cells⁴⁵.

In summary, our study provides compelling evidence at the cellular level that the Purkinje fiber network are critical contributors to arrhythmia initiation in CPVT and that defects in intracellular Ca²⁺ regulation underlie the disease phenotype. These data add to an accumulating body of literature suggesting that the unique structural and functional properties of PCs and the network they comprise play a mechanistic role in a broad range of arrhythmic syndromes. Moreover, our data provide impetus for focusing on PCs as specific anti-arrhythmic targets.

Novelty and Significance

What is known?

• Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia that is characterized by aberrant regulation of intracellular calcium handling.

• Studies at the organ or organismal level of resolution suggest that the Purkinje fiber network may serve as the arrhythmic trigger in CPVT.

What new information does this article contribute?

• We determined the kinetic properties of intracellular calcium transients in adult ventricular myocytes and Purkinje cells from both wild type and RyR2^R4496C/+ mutant mice.

• We found that both wild type and RyR2^R4496C/+ mutant Purkinjes cells have a great propensity to develop unstimulated spontaneous calcium release events and triggered action potentials compared to working ventricular myocytes of the same genotype.

• We determined that flecainide and tetracaine both potently suppress spontaneous calcium release events in Purkinje cells.

Multiple lines of evidence suggest that the cardiac Purkinje fiber network serves as the arrhythmic trigger in a range of genetic and acquired forms of heart disease. However, to date, the evidence is largely indirect, including electrical recordings from the subendocardium in the intact or isolated heart, or suppression of arrhythmias following ablation of presumptive cells from the Purkinje fiber network. Here, using a novel genetic reporter gene to specifically identify dissociated adult murine Purkinje cells, we show for the first time that Purkinje cells are indeed more likely than working ventricular myocytes to develop unstimulated spontaneous calcium release events that are capable of triggering action potentials. This preferential behavior is exacerbated in in mice harboring a CPVT disease-causing mutation in the RyR2 channel, especially so in the presence of isoproterenol. Moreover, we show that Purkinje cells can respond to anti-arrhythmic agents, but their sensitivities may differ from working ventricular myocytes. Our study provides new insight into disease pathogenesis at the cellular level of resolution, and also provides impetus for the development of Purkinje cell-specific anti-arrhythmic strategies. Such strategies may find utility both in inherited arrhythmias such as CPVT, but potentially also in acquired syndromes including post-myocardial infarction ventricular arrhythmias.

Non-standard Abbreviations and Acronyms

CPVT

Catecholaminergic polymorphic ventricular tachycardia

RYR2

ryanodine receptor type 2

CASQ2

calsequestrin

CCS

cardiac conduction system

PC

Purkinje cell

VM

ventricular myocyte

SCaE

spontaneous calcium release event