Multiphoton Imaging of Ca2+ Instability in Acute Myocardial Slices from a RyR2R2474S Murine Model of Catecholaminergic Polymorphic Ventricular Tachycardia

Abstract

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a familial stress-induced arrhythmia syndrome, mostly caused by mutations in Ryanodine receptor 2 (RyR2), the sarcoplasmic reticulum (SR) Ca²⁺ release channel in cardiomyocytes. Pathogenetic mutations lead to gain of function in the channel, causing arrhythmias by promoting diastolic spontaneous Ca²⁺ release (SCR) from the SR and delayed afterdepolarizations. While the study of Ca²⁺ dynamics in single cells from murine CPVT models has increased our understanding of the disease pathogenesis, questions remain on the mechanisms triggering the lethal arrhythmias at tissue level. Here, we combined subcellular analysis of Ca²⁺ signals in isolated cardiomyocytes and in acute thick ventricular slices of RyR2^R2474S knock-in mice, electrically paced at different rates (1-5 Hz), to identify arrhythmogenic Ca²⁺ dynamics, from the sub- to the multicellular perspective. In both models, RyR2^R2474S cardiomyocytes had increased propensity to develop SCR upon adrenergic stimulation, which manifested, in the slices, with Ca²⁺ alternans and synchronous Ca²⁺ release events in neighboring cardiomyocytes. Analysis of Ca²⁺ dynamics in multiple cells in the tissue suggests that SCRs beget SCRs in contiguous cells, overcoming the protective electrotonic myocardial coupling, and potentially generating arrhythmia triggering foci. We suggest that intercellular interactions may underscore arrhythmic propensity in CPVT hearts with 'leaky' RyR2.

Keywords: Ca2+ imaging; arrhythmias; cardiac slices; cardiomyocytes; catecholaminergic polymorphic ventricular tachycardia; ryanodine receptor 2.

Figure 1

Spontaneous Ca²⁺ release in adult cardiomyocytes isolated from CPVT mouse hearts. (A) Fluorescence image of Ca²⁺ dynamics monitored with confocal microscopy in a heterozygous RyR2^RS/wt (RS/WT) CM during isoproterenol (ISO, 1 μM) stimulation. Upon steady-state pacing, the last depolarization pulse (p) activates a regular intracellular Ca²⁺ transient, followed by abnormal spontaneous (un-paced) Ca²⁺ waves: * localized; ** cell-wide; *** regenerative. F/Fo, normalized fluorescence Ca²⁺ signal intensity. (B) Bar graph summarizing cell-wide Ca²⁺ wave occurrence in WT and CPVT CMs in basal conditions vs. ISO stimulation. Error bars represent SD; * p < 0.05.

Figure 2

Multiphoton Ca²⁺ imaging of Fluo-4-loaded myocardial slices. (A) Time course of Ca²⁺ fluctuations in a heart slice, showing rises in fluorescence intensity, represented in pseudocolor (see reference bar on the left margin of the panel) upon electrical stimulation. F/Fo, normalized fluorescence Ca²⁺ signal intensity. (B) Fluorescence intensity profile of three different cells (in A, 240 ms) showing synchronized changes during electrical field stimulation. Stimulation pulses are indicated by the red lines. (C) Quantification of Ca²⁺ transient amplitude, in both WT and CPVT heart slices, paced at 1 Hz, both in the absence (black bars) and in the presence (white bars) of norepinephrine (NE, 1 μM) (* p < 0.05 (WT vs. WT + NE); # p < 0.05 (CPVT + NE vs. WT + NE) n = 30 cells/condition). (D) Representative trace of spontaneous Ca²⁺ release events occurring between two consecutive beats (highlighted by the red box). (E) Quantification of SCRs in CPVT and WT slices, in unstimulated conditions and upon perfusion with NE (1 μM). Bars represent SD (* p < 0.05 compared to CPVT-NE; n = 40 cells for each group).

Figure 3

CPVT heart slices develop Ca²⁺ alternans upon β-AR stimulation. (A) Fraction of cells developing Ca²⁺ alternans in WT and RyR2^RS/wt slices, paced at 2 Hz, during β-AR stimulation with NE (1 μM). (B) Representative plots of Ca²⁺ oscillations in three adjacent CMs in a RyR2^RS/wt slice upon NE perfusion, showing alternans synchronous in phase, but not in amplitude (red bars indicate electrical pacing). (C) Line scan profiles from CPVT CMs, in basal (blue line) or NE-stimulated (black line) conditions, during 2 Hz stimulation. (D) Decay time of Ca²⁺ transients calculated on the line scan profiles in (C), for both WT and CPVT slices, paced at 2 Hz. Error bars represent SD (n = 25 cells/condition; ** p < 0.01 compared to WT; ## p < 0.01 compared to CPVT in basal condition).

Figure 4

Pace-stop stimulation protocol unmasks spontaneous untriggered Ca²⁺ release in CPVT slices. (A) Fluorescence profiles of CMs within cardiac slice from heterozygous RyR2^RS/wt mice, in control conditions (black trace) and during NE administration (red trace), showing a representative trace of a SCR. (B) Quantification of SCRs in the slices from WT and CPVT hearts at baseline and in the presence of NE. Error bars represent SD (** p < 0.01 compared to WT plus NE; # p < 0.001 compared to CPVT in basal condition). (C) WT and CPVT slices showed significantly different latency time before the first spontaneous Ca²⁺ release event. Error bars represent SD (* p < 0.05 compared to WT; ** p < 0.01 compared to WT; # p < 0.05 CPVT in basal condition vs. NE administration).

Figure 5

Diastolic SCR synchronization in myocardial cell clusters. (A) Cells in CPVT slices showed significantly shorter latency, compared to controls (WT), between the first SCR upon pacing interruption and the subsequent ones in neighboring CMs. Error bars represent SD (* p < 0.05 compared to WT in basal condition; # p < 0.01 compared to CPVT upon NE stimulation). (B) Representative traces of Ca²⁺ dynamics in a CPVT slice that underwent a pace-stop protocol. The colored traces show intracellular Ca²⁺ changes in the cells identified by the same color in the regions of interest (ROIs) delineated in the corresponding image on the right side of the panel. The rectangles above the chart highlight, in the same color coding, the duration of spontaneous Ca²⁺ elevations, defined as increase of the fluorescence signal higher than two SDs of the basal fluorescence intensity. Arrows connect the individual boxes, each one representing a single cell in the field, showing the intercellular transmission of temporally overlapped SCRs in adjacent cells, which underscores the influence SCR may have on further SCRs. In the representative example shown, the initiating event occurs in cell #3 (orange line and box) and is subsequently transmitted to the others.

Figure 6

Factors potentially contributing to synchronization of diastolic depolarization in the myocardium. A “source–sink–source” model, showing the effect of a SCR occurring in one myocardial excitable cell (red labeled “source” cell) on vulnerability of surrounding CM to develop, in turn, diastolic “SR leak” and SCR. This positive feedback loop may, thus, “regenerate” a current source, enlarging the area of CMs depolarizing synchronously.