Constitutive Intracellular Na+ Excess in Purkinje Cells Promotes Arrhythmogenesis at Lower Levels of Stress Than Ventricular Myocytes From Mice With Catecholaminergic Polymorphic Ventricular Tachycardia

Abstract

Background: In catecholaminergic polymorphic ventricular tachycardia (CPVT), cardiac Purkinje cells (PCs) appear more susceptible to Ca(2+) dysfunction than ventricular myocytes (VMs). The underlying mechanisms remain unknown. Using a CPVT mouse (RyR2(R4496C+/Cx40eGFP)), we tested whether PC intracellular Ca(2+) ([Ca(2+)]i) dysregulation results from a constitutive [Na(+)]i surplus relative to VMs.

Methods and results: Simultaneous optical mapping of voltage and [Ca(2+)]i in CPVT hearts showed that spontaneous Ca(2+) release preceded pacing-induced triggered activity at subendocardial PCs. On simultaneous current-clamp and Ca(2+) imaging, early and delayed afterdepolarizations trailed spontaneous Ca(2+) release and were more frequent in CPVT PCs than CPVT VMs. As a result of increased activity of mutant ryanodine receptor type 2 channels, sarcoplasmic reticulum Ca(2+) load, measured by caffeine-induced Ca(2+) transients, was lower in CPVT VMs and PCs than respective controls, and sarcoplasmic reticulum fractional release was greater in both CPVT PCs and VMs than respective controls. [Na(+)]i was higher in both control and CPVT PCs than VMs, whereas the density of the Na(+)/Ca(2+) exchanger current was not different between PCs and VMs. Computer simulations using a PC model predicted that the elevated [Na(+)]i of PCs promoted delayed afterdepolarizations, which were always preceded by spontaneous Ca(2+) release events from hyperactive ryanodine receptor type 2 channels. Increasing [Na(+)]i monotonically increased delayed afterdepolarization frequency. Confocal imaging experiments showed that postpacing Ca(2+) spark frequency was highest in intact CPVT PCs, but such differences were reversed on saponin-induced membrane permeabilization, indicating that differences in [Na(+)]i played a central role.

Conclusions: In CPVT mice, the constitutive [Na(+)]i excess of PCs promotes triggered activity and arrhythmogenesis at lower levels of stress than VMs.

Keywords: arrhythmias, cardiac; calcium; calcium signaling; sodium-calcium exchanger.

Figure 1.

Subendocardial origins of spontaneous beats. A, Volume-conducted ECG recording of a catecholaminergic polymorphic ventricular tachycardia heart with 3.6 mmol/L Ca²⁺ and 160 nmol/L isoproterenol before, during, and after 12-Hz burst pacing. Nine abnormal action potentials (APs) were induced after pacing (red box, numbered). B, Earliest right ventricular endocardial AP wave breakthroughs of abnormal beats (1–9, each in a separate box) shown by white and magenta spots (color index bar represents percent from maximal signal in each frame). C, Image of green fluorescent protein–mapped area of the Purkinje network (left) superimposed with white contour lines from B (middle and right). Note colocalization of triggered beats with branches of the Purkinje conduction system.

Figure 2.

Spontaneous Ca²⁺ release precedes voltage depolarization during triggered activity in the subendocardium of the catecholaminergic polymorphic ventricular tachycardia heart. The heart was perfused with 3.6 mmol/L Ca²⁺ and 160 nmol/L isoproterenol. A, Volume-conducted ECG (top, blue) and optical signals obtained from a single pixel (black, voltage; red, Ca²⁺) at the earliest breakthrough on a subendocardial Purkinje fiber location. B, Enlarged representative traces for sinus (left) and triggered (right) beats. Symbols on top identify the specific beats selected from A. C, Beat-to-beat (left) and mean (right) Vm-Ca²⁺ timing difference maps for sinus (top), triggered (middle), and recovery (bottom) beats measured at 60% maximum depolarization (horizontal black lines in B).

Figure 3.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) Purkinje cells (PCs) have lower threshold for Ca²⁺-linked triggered activity. Representative simultaneous action potential (black) and Ca²⁺ fluorescence (red) recordings are presented on the same time scale. A and B, Steady-state 1-Hz pacing traces at baseline (left) and after 30 nmol/L isoproterenol (ISO) perfusion (right) for CPVT ventricular myocytes (VMs) and CPVT PCs, respectively. C and D, Steady-state 5-Hz pacing traces at baseline (left) and after 30 nmol/L isoproterenol perfusion (right) for CPVT VMs and CPVT PCs, respectively. Addition of isoproterenol significantly increases Ca²⁺ transient amplitude in both cell types. Neither control VMs nor control PCs developed abnormal beats under the same conditions (not shown). Black arrows indicate paced beats; blue arrows, spontaneous triggered activity.

Figure 4.

Ca²⁺ spark frequency (CaSpF) is higher in control and catecholaminergic polymorphic ventricular tachycardia (CPVT) Purkinje cells (PCs) than in ventricular myocytes (VMs). A, Three-dimensional surface plots of representative line scan images for a CPVT VM and CPVT PC for baseline (left) and 10 nM/L isoproterenol (right). B, Mean CaSpF at 1-, 3-, and 5-Hz stimulation. *P<0.05 vs control VMs at the same pacing frequency. #P<0.05 vs all other cell groups at the same pacing frequency. C, Time course of responses of CPVT VMs and CPVT PCs to isoproterenol (ISO; 10 nmol/L). *P<0.05 in Purkinje CPVT myocytes vs all other cell groups at same time point after isoproterenol.

Figure 5.

Sarcoplasmic reticulum (SR) Ca²⁺ handling and high [Na⁺]_i in Purkinje cells (PCs). A, Representative traces of caffeine-induced Ca²⁺ transient from control ventricular myocytes (VMs; top left) and PCs (bottom left) and catecholaminergic polymorphic ventricular tachycardia VMs (top right) and PCs (bottom right) recorded at 3-Hz stimulation with 10 mmol/L caffeine. B, Mean peak SR Ca²⁺ content in each cell type. C, SR fractional release and (D) time to 50% decay from caffeine-induced Ca²⁺ transient VMs. E, Average NiCl₂-sensitive Na⁺/Ca²⁺ exchanger current/voltage relationships (voltage ramp protocol shown in the inset). F, Mean [Na⁺] concentration. *P<0.05.

Figure 6.

Pacing induced delayed afterdepolarizations (DADs) and triggered activity (TA) in the numeric mouse Purkinje cell model with spontaneous Ca²⁺ release enabled (catecholaminergic polymorphic ventricular tachycardia model; A) control case and (B) in the presence of isoproterenol effects followed by burst pacing at 5 Hz.

Figure 7.

Ca²⁺-linked arrhythmogenesis in the [Na]_i-overloaded numeric Purkinje cell model with isoproterenol effects. A, Generation of frequent delayed afterdepolarizations and triggered activity in the catecholaminergic polymorphic ventricular tachycardia model at various elevated levels of [Na⁺]_i obtained by reducing I_NaK density. B, Corresponding Ca²⁺ release flux from the ryanodine channels shows large-magnitude spontaneous Ca²⁺ release events preceding the membrane depolarizations.

Figure 8.

Membrane permeabilization terminates differential Ca²⁺ spark frequency (CaSpF) in Purkinje cells (PCs) compared with ventricular myocytes (VMs). A through D, Representative confocal line scan recordings showing Ca²⁺ sparks under 50 nmol/L free Ca²⁺ in permeabilized control VMs, control PCs, catecholaminergic polymorphic ventricular tachycardia (CPVT) VMs, and CPVT PCs. E, Average CaSpF among all groups. Note that there is no significant difference in CaSpF between control VMs and control PCs and that there is a significant decrease in CaSpF between CPVT VMs and CPVT PCs. *P<0.05.