Total internal reflectance fluorescence imaging of genetically engineered ryanodine receptor-targeted Ca2+ probes in rat ventricular myocytes

Abstract

The details of cardiac Ca²⁺ signaling within the dyadic junction remain unclear because of limitations in rapid spatial imaging techniques, and availability of Ca²⁺ probes localized to dyadic junctions. To critically monitor ryanodine receptors' (RyR2) Ca²⁺ nano-domains, we combined the use of genetically engineered RyR2-targeted pericam probes, (FKBP-YCaMP, K_d=150nM, or FKBP-GCaMP6, K_d=240nM) with rapid total internal reflectance fluorescence (TIRF) microscopy (resolution, ∼80nm). The punctate z-line patterns of FKBP,²-targeted probes overlapped those of RyR2 antibodies and sharply contrasted to the images of probes targeted to sarcoplasmic reticulum (SERCA2a/PLB), or cytosolic Fluo-4 images. FKBP-YCaMP signals were too small (∼20%) and too slow (2-3s) to detect Ca²⁺ sparks, but the probe was effective in marking where Fluo-4 Ca²⁺ sparks developed. FKBP-GCaMP6, on the other hand, produced rapidly decaying Ca²⁺ signals that: a) had faster kinetics and activated synchronous with I_Ca³ but were of variable size at different z-lines and b) were accompanied by spatially confined spontaneous Ca²⁺ sparks, originating from a subset of eager sites. The frequency of spontaneously occurring sparks was lower in FKBP-GCaMP6 infected myocytes as compared to Fluo-4 dialyzed myocytes, but isoproterenol enhanced their frequency more effectively than in Fluo-4 dialyzed cells. Nevertheless, isoproterenol failed to dissociate FKBP-GCaMP6 from the z-lines. The data suggests that FKBP-GCaMP6 binds predominantly to junctional RyR2s and has sufficient on-rate efficiency as to monitor the released Ca²⁺ in individual dyadic clefts, and supports the idea that β-adrenergic agonists may modulate the stabilizing effects of native FKBP on RyR2.

Keywords: Ca(2+) sparks; Cardiac Ca(2+) signaling; Dyadic Ca(2+)signals; FKBP; Genetically engineered Ca(2+) probe; Ryanodine receptor.

Figure 1. Two new fluorescent probes targeted to ryanodine receptors with FKBP12.6 (A) and confocal images of their distribution in adult rat ventricular cells (B-C) as compared to probes targeted to SERCA (D) and mitochondria (E)

A: Schematic cDNA sequences for two new probes, FKBP-YCamP and FKBP-GCaMP6, where FKBP12.6 is linked to the N terminal of previously used constructs (Based on[13, 15, 22, 23]). B: Contrast–enhanced 3-D reconstruction based on z-stack showing targeting of FKBP-YCaMP to z-lines. C-E: Comparison confocal images showing the distribution of fluorescent probes targeted with FKBP to RyR (C: FKBP-YCaMP), phospholamban to SERCA (D: PLN-YCaMP) and the mitochondrial targeting sequence of subunit VIII of human cytochrome c oxidase (E: Mitycam[13, 14]). The confocal plane was adjusted to the midline of the cells where blue DAPI staining shows the locations of nuclei in the fixed cells in panels C and D, but not in the live cell in panel E. Inset panels show enlargements of boxed areas.

Figure 2. Colocalization of FKBP-YCaMP (A) and FKBP-GCaMP6 (B) with immunolabeled RyR (RyR2 antibody)

Figure 3. TIRF imaging clearly resolved sarcomeric fluorescence patterns of fluorescent Ca²⁺ probes targeted to RyR (A) and mitochondria (B) in strongly adhering ventricular cardiomyocytes (C, D)

E: Changes in fluorescence intensity vs. [Ca²⁺] in permeabilized, FKBP-YCaMP expressing cell.

Figure 4. Comparison of cellular Ca²⁺ transients measured simultaneously with FKBP-YCaMP and Fura-2

A: Images showing decreases in the local intensity of FKBP-YCaMP measured on activation of I_Ca by depolarization from -50 mV to 0 mV and selected regions of interest (ROI). B: Time course of fluorescence changes in ROIs and (expanded) current traces indicating timing of activation of I_Ca. C: Images of Fura-2 fluorescence in cardiomyocyte showing uniform decreases in intensity during caffeine triggered Ca²⁺ release. D: Interlaced recording of Ca²⁺ signals measured with FKBP-YCaMP and Fura-2 showing slower rise (histogram) and decay times for the genetically engineered probe.

Figure 5. Ca²⁺ sparks measured with fluo-4 in cells with and without expression of FKBP-YCaMP

A: Baseline fluorescence of FKBP-YCaMP and Fluo-4 both excited at 488 nm. B: Contrast enhanced image showing targeting of FKBP-YCaMP to z-lines. C: Detail of panel B with color-coded regions of interest (ROI) and numbered sample frames showing examples of infrequent (top) or regularly recurring Fluo-4 Ca²⁺ sparks (bottom, red and orange ROIs). D: Time course of fluorescence intensity measured at 30 Hz in ROIs with details shown on expanded time scale in panel H. Panels E, F and G: Comparison of the frequency (E), duration (F), from recordings at ∼100 Hz and amplitude (G) of Fluo-4 Ca²⁺ sparks measured with Fluo-4 in control cells and in cells expressing FKBP-YCaMP.

Figure 6. Comparison of local I_Ca-activated Ca²⁺ signals measured with FKBP-GCaMP6 (A, B, C) and FKBP-YCaMP (D, E, F)

A, D: Contrast enhanced images of background fluorescence measured with FKBP-GCaMP6 (A) and FKBP-YCaMP (D) and color-coded regions of interest (ROI) defined along sarcomeric z-line ridges. B, E: Time course of I_Ca–activated fluorescence changes (ΔF/F₀) measured in 3 selected ROIs, preceded by columns comparing their peak amplitude. C, F: Variability in ΔF/F₀ measured at a sequence of sarcomeric lines as defined in panes A and D. The symbols in panels C and D correspond to 5 different cells for each of the FKBP-linked probes.

Figure 7. Properties of Ca²⁺ sparks measured with GCaMP6-FKBP (A) and Fluo-4 (C)

A, E: Background fluorescence and sample frames with Ca²⁺ sparks recorded in different cells. B: Time course of fluorescence intensity at the location showing Ca²⁺ sparks in panels A (top, FKBP-GCaMP6) and C (bottom, Fluo-4). D: Average values of Ca²⁺ spark parameters (amplitude, frequency of occurrence, rise time) measured with FKBP-GCaMP6 (green columns) and Fluo-4 (black columns). E, F: Two sequences of fluorescence images showing background fluorescence (F₀ with, E, and without, F, contrast enhancement) followed by sequences of ΔF-images recorded at 100 Hz.

Figure 8. Effect of isoproterenol on number of Ca²⁺ sparks measured with Fluo-4 in control cells and in cells expressing FKBP-GCaMP6

A: Sample frames selected to illustrate the increase in the number of Ca²⁺ sparks that is produced by 1 μM isoproterenol in cells expressing FKBP-GCaMP6. B: Histogram showing average values of Ca²⁺ spark frequency.