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 Ca(2+) 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 Ca(2+) release events and the response to the antiarrhythmic 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 ventricular myocytes (EGFP(-)) and Purkinje cells (EGFP(+)) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) 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 tau(decay) in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2(R4496C/+) mutant Purkinje cells were also most likely to develop spontaneous Ca(2+) release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca(2+(i)) events, the RyR2(R4496C/+) Purkinje cells responded with the most profound abnormalities in intracellular Ca(2+) handling, including a significant increase in the frequency of unstimulated Ca(2+(i)) events and 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 Ca(2+) 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 Ca(2+) handling than ventricular myocytes. This proarrhythmic behavior is enhanced by disease-causing mutations in the RyR2 Ca(2+) 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.

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.

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.

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.

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.

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.