Solute movement in the t-tubule system of rabbit and mouse cardiomyocytes

Figures

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  Fig. 1.. Steady-state solute penetrance of t-tubules in rabbit and mouse ventricular cardiomyocytes. (A) Fluorescence images of rabbit (Left) and mouse (Right) cardiomyocytes bathed in a 10-kDa rhodamine B-linked dextran reference dye (Top) and 4- or 70-kDa fluorescein-linked dextran test dyes (Middle). Cells were incubated for >10 min to reach steady state. The test-to-reference ratio (FT/FR) images are shown (Bottom) (color table at the right). (Scale bar for all panels: 5 μm.) (B) Stokes radii of the test solutes, as measured with FCS, and compared with MW1/3. Error bars for three to six determinations were generally smaller than the symbol size. Open symbols indicate dextran-based probes. (C) Dependence of FT/FR from rabbit (red circles) and mouse (blue crosses) on the measured Stokes radius. Numbers of independent determinations are indicated beside each data point.

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  Fig. 2.. Effect of bath superfusion on FRAP. (A) Photobleaching profile (x–z) from a 1.2 N.A. water immersion objective. (Scale bar: 5 μm.) (B) A part of the computational space showing position of cell above coverslip (at arrow) and limits of the FRAP illumination cone (produced by the objective N.A.) as well as the nonlinear mesh used for solving equations. (C) Calculation of local flow velocity above coverslip. Color scale at the top shows fraction of bulk flow velocity (2 mm⋅s−1). A magnified region (white dashed box) surrounding the model is shown (Right) with flow velocities displayed on a log scale from 0.3 mm⋅s−1 to 0.1 μm⋅s−1 with colors as indicated at the top. (D) Simulation of FRAP at a bulk superfusion flow rate of 1 mm⋅s−1 after the end of a 5-s bleach period (t = 0), and at t = 1 and t = 2 s. Diffusion coefficient of solute was assumed to be 1.45 × 10−10 m2⋅s−1 for a 3-kDa dextran with diffusion inside the cell reduced by a factor of 22 (7). (Scale bars in B–D: 20 μm.) (E) FRAP inside the modeled cell at varying superfusion flow rates, from 0 to 2 mm⋅s−1, as well as with no extracellular depletion effects.

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  Fig. 3.. Kinetics of solute entry into t-tubules examined by FRAP with no solute flow. (A) Exemplar fluorescence images of rabbit and mouse cardiomyocytes in the ∼1-kDa test solute. The FRAP bleach region (green) and region of interest where recovery was tracked (red for rabbit, blue for mouse) are shown. (Scale bars: 10 μm.) (B) FRAP time course for a rabbit (red) and mouse (blue) cardiomyocytes. (C) Time for 20%, 50%, and 63% recovery. **P < 0.01; ****P < 0.001, two-way ANOVA with Bonferroni post hoc analysis. (D) Depth of photobleaching during FRAP. *P < 0.05, Mann–Whitney U test. (E) Length, width, and depth of cardiomyocytes used for FRAP. ****P < 0.001, for rabbit vs. mouse Mann–Whitney U test. (F) FRAP T50 as a function of cell depth (circles) or width (triangles) as measured from confocal z-sections. For C–F, red indicates rabbit (n = 18) and blue mouse (n = 18) cardiomyocytes. (G) Calculated change in T50 as a function of cell cross-section ellipticity (width/depth; illustrated by cartoons). Note that cells do not always orient conveniently on the coverslip so that both cell width (in x–y) and cell depth (in z) need to be measured.

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  Fig. 4.. T-tubule morphology in rabbit and mouse ventricular cardiomyocytes. (A) Confocal images of rabbit (Left) and mouse (Right) t-tubules labeled with di-8-ANEPPS (Top) and corresponding binary images (Bottom). For illustrative purposes, the rabbit and mouse t-tubule diameters are set to their means of 360 and 170 nm (respectively) (16). (Scale bar: 5 μm.) (B) Proportion of 6- × 6-μm quadrants that are internally connected in the directions shown for rabbit (n = 20 quadrants, 6 cells) and mouse (n = 48 quadrants, 5 cells; **P < 0.01, two-way ANOVA with Bonferroni post hoc analysis). (C) Branch-point density of t-tubule network. **P < 0.01, Mann–Whitney U test. (D) Tortuosity (ζ) of quadrants calculated by TauFactor (23). ***P < 0.005, two-way ANOVA with Bonferroni post hoc test. (E) Local variations in t-tubule width measured using a shape-based analysis method (16). The colors indicate the local width in micrometers with the width color scale at the right. (Scale bar: 2 µm.) (F) Distribution of t-tubule widths in living rabbit (n = 11) and mouse (n = 12) cardiomyocytes (total t-tubule length analyzed was ∼1 mm). The continuous curve was randomly sampled for simulations. (G) Effect of…

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  Fig. 5.. Computational modeling of FRAP in rabbit (Left) and mouse (Right) cardiomyocytes, incorporating measured cell dimensions, t-tubule distribution, and illumination geometry. (A) Solute concentration at t = 0 and 2 s after photobleaching. (Scale bar: 20 μm.) (B) FRAP time course with model fit and residual errors (Bottom). (C) Spatial fluorescence profiles at t = 0 s, corresponding model fits and residual errors (Bottom). For B and C, red lines show model fits when t-tubule Dapp was uniform across the cell. Blue lines show fits incorporating a thin (0.2 μm) region of slowed diffusion (reduced to 1.5% of the free t-tubule diffusion coefficient) at the t-tubule mouth.

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  Fig. 6.. Electron microscopy and tomography of rabbit (Left) and mouse (Right) cardiomyocyte t-tubule at the cell surface. (A) Electron micrographs of t-tubule entrances (indicated by red arrows). Note the basement membrane and fuzzy ground substance. (B) Electron tomograms of t-tubule entrances with corresponding 3D reconstructions of the ground substance. Note that for rabbit (red) the ground substance appears to leave a visible lumen (red arrow, Bottom), while in mouse (blue) the ground substance appears more uniform across the entire width of the lumen.

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  Fig. 7.. Relative contribution of t-tubule geometric factors on solute movement within the t-tubule system for rabbit (red bars) and mouse (blue bars) ventricular cardiomyocytes. Bars show fold reduction in Dapp (from left to right) as calculated from FRAP experiments in rabbit (n = 5) and mouse (n = 5) cardiomyocytes and reduction explainable by (i) an increase in path length due to tortuosity, (ii) the contribution from longitudinal t-tubules, (iii) variations in tubule width (varicosities), and (iv) a thin barrier of slowed diffusion in the t-tubule entrances near the cell surface. The rightmost set of bars show the product of these four t-tubule geometric factors, representing their combined effect. The shaded areas link the observed values (leftmost set of bars) to the product of factors to indicate their similarity.