Detection of Ca2+ transients near ryanodine receptors by targeting fluorescent Ca2+ sensors to the triad

Figures

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  Figure 1.. Expression pattern and Ca2+ sensitivity of GCaMP6f and T306-GCaMP6f. (A) Illustrative examples of the global and patchy expression patterns of GCaMP6f (left) and T306-GCaMP6f (right) in muscle fibers, respectively. (B) Typical subcellular fluorescence pattern of GCaMP6f (left) and T306-GCaMP6f (right) in muscle fibers. (C) Fluorescence profile along the main axis of the muscle fibers within the box-highlighted region in the above-corresponding image in B. The y scale corresponds to the background-corrected fluorescence intensity normalized by the mean intensity of the profile. (D) Confocal frames from a muscle fiber coexpressing T306-GCaMP6f (green channel) and mRFP-tagged junctophilin 1 (red channel). The right panel shows the overlay. (E) Confocal fluorescence images from a T306-GCaMP6f expressing muscle fiber upon equilibration with solutions of varying pCa (−log [Ca2+]; above indicated values). (F) Ca2+ dependence of the relative change in fluorescence in three fibers expressing T306-GCaMP6f (red filled squares and triangles), one fiber expressing GCaMP6f (open blue circles), and droplets of calibrating solutions containing rhod-2 (filled black circles). Superimposed colored…

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  Figure S1.. Voxel map presenting the distribution of the spatial positions used for simulating the changes in Ca2+ and Ca2+ bound to GCaMP6f or T306-GCaMP6f along half a sarcomere following a synthetic input Ca2+ release flux occurring at the triadic junction. Ni corresponds to the number of voxels at each position (see Materials and methods).

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  Figure 2.. Kinetic features of GCaMP6f Ca2+ transients. (A) Simultaneous GCaMP6f (thick blue traces) and rhod-2 (thin black traces) fluorescence transients detected from a muscle fiber in response to the voltage-clamp protocol shown on top, consisting of pulses to +60 mV of 5, 10, 20, and 50 ms duration. The respective GCaMP6f and rhod-2 fluorescence transients are shown scaled to a similar peak amplitude, with corresponding blue and black y scale bars both indicating a fluorescence change of 2 × F0. The inset on the right shows the kinetically corrected Ca2+ transients calculated from the GCaMP6f and rhod-2 traces elicited by the 5-ms-long pulse and by the 20-ms-long pulse (see text for details). The two graphs at the bottom report mean values for the time to peak F/F0 and for the time constant of F/F0 decay (τdecay) after repolarization, from three fibers expressing GCaMP6f, loaded with rhod-2, and stimulated with the same voltage-clamp protocol. Filled black circles and open blue circles correspond to measurements from rhod-2 and GCaMP6f transients, respectively. Vertical bars represent the SEM. (B) Illustrative example of simultaneously detected GCaMP6f (thick blue traces) and rhod-2…

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  Figure 3.. Kinetic features of T306-GCaMP6f Ca2+ transients elicited by short depolarizing pulses. Simultaneous T306-GCaMP6f (thick red traces) and rhod-2 (thin black traces) fluorescence transients detected from a muscle fiber in response to the depolarizing pulse protocol shown on top. The respective T306-GCaMP6f and rhod-2 fluorescence transients are shown scaled to a similar peak amplitude, with corresponding red and black y scale bars both indicating a fluorescence change of 0.5 × F0. The two graphs at the bottom report mean values for the time to peak F/F0 and for the time constant of F/F0 decay (τdecay) after repolarization, from four fibers expressing T306-GCaMP6f, loaded with rhod-2, and stimulated with the same voltage-clamp protocol. Filled black circles and open red circles correspond to measurements from rhod-2 and T306-GCaMP6f transients, respectively. Vertical bars represent the SEM.

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  Figure 4.. Ca2+ transients within a muscle fiber region exhibiting a gradient of T306-GCaMP6f expression. (A) T306-GCaMP6f (red trace) and rhod-2 (black trace) distribution along a line scanned in a fiber region exhibiting a T306-GCaMP6f gradient of expression. (B) Voltage-activated T306-GCaMP6f and rhod-2 transients recorded within the high- (red and black traces in the left and right panel, respectively) and low-expression region (superimposed gray traces) of T306-GCaMP6f. The high- and low-expression line regions over which fluorescence transients were averaged are indicated by horizontal colored (red or black) and gray lines, respectively, at the bottom of the fluorescence distribution profiles in A. AU, arbitrary units.

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  Figure 5.. Voltage-dependent GCaMP6f and T306-GCaMP6f Ca2+ transients. (A) Illustrative examples of fluorescence transients from a muscle fiber expressing GCaMP6f (left) and a fiber expressing T306-GCaMP6f (right) in response to 500-ms-long depolarizing pulses of increasing amplitude. The curves superimposed to the decay of the transients correspond to the result from fitting a single exponential to the decay phase. The inset in each panel shows the average trace in response to a pulse to −15 mV from seven fibers expressing GCaMP6f (left) and six fibers expressing T306-GCaMP6f (right). (B) Mean voltage dependence of GCaMP6f (open blue circles) and T306-GCaMP6f (open red circles) fluorescence transients F/F0 peak amplitude (Peak F/F0), time to 80% peak amplitude (T80%), and time constant of decay (τdecay). Mean values at the different voltage levels are from 7–14 fibers for GCaMP6f and 3–11 fibers for T306-GCaMP6f. Vertical bars represent the SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

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  Figure 6.. Model simulation of the spatial gradient of Ca2+ and of calcium bound to the GCaMP6f probes along a 0.5-µm distance from the Ca2+ release site following a low level of SR Ca2+ release activation. (A) Synthetic Ca2+ release input flux. (B and C) Spatial gradient of Ca2+ change (B) and corresponding Ca2+ bound to GCaMP6f (C). (D and E) Spatial gradient of Ca2+ change (D) and corresponding Ca2+ bound to T306-GCaMP6f (E). In B–E, traces underneath the image show the time course of change in the given parameter at the triad level (thick red trace), at the mid-sarcomere level (thin blue trace), and the average change throughout the distance (dotted black trace).

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  Figure 7.. Model simulation of the spatial gradient of Ca2+ and of calcium bound to the GCaMP6f probes along a 0.5-µm distance from the Ca2+ release site following a large level of SR Ca2+ release activation. (A) Synthetic Ca2+ release input flux. (B and C) Spatial gradient of Ca2+ change (B) and corresponding Ca2+ bound to GCaMP6f (C). (D and E) Spatial gradient of Ca2+ change (D) and corresponding Ca2+ bound to T306-GCaMP6f (E). In B–E, traces underneath the image show the time course of change in the given parameter at the triad level (thick red trace), at the mid-sarcomere level (thin blue trace), and the average change throughout the entire distance (dotted black trace).

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  Figure 8.. Model simulation of the average changes in Ca2+ bound to GCaMP6f and to T306-GCaMP6f following different levels of SR Ca2+ release activation. (A) Superimposed synthetic Ca2+ release input fluxes used for the simulation (1–4). (B) Calculated average changes in Ca2+ bound to GCaMP6f (left, blue) or T306-GCaMP6f (right, red) following each of the four Ca2+ release input fluxes shown in A. (C) Normalized time course of the calculated average changes in Ca2+ bound to GCaMP6f (continuous blue traces) and T306-GCaMP6f (dotted red traces) in response to the Ca2+ release fluxes shown in A. Changes in Ca2+ bound to each of the two probes for a same given input flux are shown superimposed. (D) Dependence upon the input flux (1–4) of the time to reach 80% of the peak change in Ca2+ bound to GCaMP6f (left, open blue circles) and T306-GCaMP6f (filled red circles) and corresponding values for the time constant of decay (right) after the end of the release flux.

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  Figure S2.. Model simulation of the spatial gradient of Ca2+ and of calcium bound to the GCaMP6f probes along a 0.5-µm distance from the Ca2+ release site following a low level of SR Ca2+ release activation, assuming a low Ca2+ diffusion coefficient. (A) Synthetic Ca2+ release input flux. (B and C) Spatial gradient of Ca2+ change (B) and corresponding Ca2+ bound to GCaMP6f (C). (D and E) Spatial gradient of Ca2+ change (D) and corresponding Ca2+ bound to T306-GCaMP6f (E). In B–E, traces underneath the image show the time course of change in the given parameter at the triad level (thick red trace), at the mid-sarcomere level (thin blue trace), and the average change throughout the distance (dotted black trace).

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  Figure S3.. Model simulation of the spatial gradient of Ca2+ and calcium bound to the GCaMP6f probes along a 0.5-µm distance from the Ca2+ release site following a large level of SR Ca2+ release activation, assuming a low Ca2+ diffusion coefficient. (A) Synthetic Ca2+ release input flux. (B and C) Spatial gradient of Ca2+ change (B) and corresponding Ca2+ bound to GCaMP6f (C). (D and E) Spatial gradient of Ca2+ change (D) and corresponding Ca2+ bound to T306-GCaMP6f (E). In B–E, traces underneath the image show the time course of change in the given parameter at the triad level (thick red trace), at the mid-sarcomere level (thin blue trace), and the average change throughout the entire distance (dotted black trace).

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  Figure S4.. Model simulation of the average changes in Ca2+ bound to GCaMP6f and T306-GCaMP6f following different levels of SR Ca2+ release activation, assuming a low Ca2+ diffusion coefficient. (A) Superimposed synthetic Ca2+ release input fluxes used for the simulation (1–4). (B) Calculated average changes in Ca2+ bound to GCaMP6f (left, blue) or T306-GCaMP6f (right, red) following each of the four Ca2+ release input fluxes shown in A. (C) Normalized time course of the calculated average changes in Ca2+ bound to GCaMP6f (continuous blue traces) and T306-GCaMP6f (dotted red traces) in response to the Ca2+ release fluxes shown in A. Changes in Ca2+ bound to each of the two probes for a same given input flux are shown superimposed. (D) Dependence upon the input flux (1–4) of the time to reach 80% of the peak change in Ca2+ bound to GCaMP6f (left, open blue circles) and T306-GCaMP6f (filled red circles) and corresponding values for the time constant of decay (right) after the end of the release flux.