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

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 RyR2RS/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 RyR2RS/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 RyR2RS/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 RyR2RS/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…

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.