Analysis of molecular movement reveals latticelike obstructions to diffusion in heart muscle cells

Abstract

Intracellular diffusion in muscle cells is known to be restricted. Although characteristics and localization of these restrictions is yet to be elucidated, it has been established that ischemia-reperfusion injury reduces the overall diffusion restriction. Here we apply an extended version of raster image correlation spectroscopy to determine directional anisotropy and coefficients of diffusion in rat cardiomyocytes. Our experimental results indicate that diffusion of a smaller molecule (1127 MW fluorescently labeled ATTO633-ATP) is restricted more than that of a larger one (10,000 MW Alexa647-dextran), when comparing diffusion in cardiomyocytes to that in solution. We attempt to provide a resolution to this counterintuitive result by applying a quantitative stochastic model of diffusion. Modeling results suggest the presence of periodic intracellular barriers situated ∼1 μm apart having very low permeabilities and a small effect of molecular crowding in volumes between the barriers. Such intracellular structuring could restrict diffusion of molecules of energy metabolism, reactive oxygen species, and apoptotic signals, enacting a significant role in normally functioning cardiomyocytes as well as in pathological conditions of the heart.

Figure 1

Diffusion of ATTO633-ATP in rat CM analyzed by RICS. In the beginning of the experiment, intact rat CMs labeled with Mitotracker Green are positioned into solution containing ATTO633-ATP (confocal images, A and B). Because cells are intact, ATTO633-ATP does not penetrate sarcolemma and fluorescence is recorded in solution surrounding the cells as well as T-tubules (B). Holes are introduced into sarcolemma by poking one of the cells with a glass pipette (transmission image of approaching pipette in C). As a result, ATTO633-ATP is able to diffuse into the cell (E) while the structure of the cell is intact (D). RICS analysis is presented in F–I. Experimental data (points) acquired at different laser scanning frequencies and directions are fitted by a model (lines). Spatial and temporal components of the correlation function are shown in panels F and G, respectively. On panels F and G, imaging was performed with the laser scanning along a line parallel to axis x at different frequencies (frequencies noted in legend on the right bottom). Correlation along the same line (main graphs of F and G); correlation of the signal between pixels in adjacent lines (insets). Due to the laser backtracking and variation in scan frequencies, temporal component has gaps visible (inset of panel G). Due to asymmetry of PSF, laser scanning in different directions leads to modification of correlation function between pixels in the same line (H) and in the adjacent lines (I). Note how the slow component leads to significant correlation in pixels in adjacent lines (I and insets of F and G).

Figure 2

Analysis of diffusion in the cell using the stochastic model. (A) Scheme of the computational model. Intracellular structure of the cell (top left) is approximated by a three-dimensional lattice of barriers that hinder molecule diffusion (top right). Barriers are placed, depending on direction α, d_α μm apart and have permeabilities p_α. Diffusion coefficient in the space between barriers is reduced by a factor λ compared to solution $\left(0<\lambda \le 1\right)$. Stochastically diffusing molecules interact with barriers and have a probability p_α of passing through (bottom left). Permeable barriers correspond to porous walls with η-pores of radius R per μm² of barrier area (bottom right). Apparent diffusion coefficients are estimated over the entire lattice. (B) Apparent diffusion constant values for ATTO633-ATP are obtained from simulations with varying barrier distances (horizontal axis) and permeabilities (indicated by values on curves). (Horizontal solid line) ATTO633-ATP diffusion coefficient estimated from experiment $\left(D_{TR}^{exp}\right)$. (Inset) Region 0.5 … 1 μm, where curves intersect with experimental data. (Triangles) Intersection points. (C) Points of intersection from panel B with permeability converted to pores per μm² for different pore radius values (10, 15, and 20 nm). Intersections of ATTO633-ATP (open triangles) and Alexa647-dextran 10K (open circles) curves of identical pore radius values signify points where model and experiment coincide for both molecules simultaneously (solid squares). Intersections are curves in three-dimensional space (barrier-to-barrier distance versus pore radius versus pores per μm²). (D) View of intersection curves from panel C in barrier-to-barrier distance and pores per μm² axes. (Lines) Different ${\lambda }_{TR}^{AATP}$ and ${\lambda }_{TR}^{ADEX}$ values. (Squares) Example intersection points from panel C. (E) Same as panel D, but for pore radius and pores per μm² values. (F) Same as panel D, but for barrier-to-barrier distance and pore radius values. AATP and ADEX represent ATTO633-ATP and Alexa647-dextran 10K, respectively.

Figure 3

Internal structure of the cardiac muscle cell. A regular arrangement of intracellular structures and organelles is present (30,55,34). Mitochondria are separated 1.8 μm in the transverse direction and 1.0 or 1.8 μm (depending on whether there are one or two mitochondria per sarcomere) in the longitudinal direction (9). Sarcomere Z lines are separated by 1.9 μm in the longitudinal direction identically with T-tubules, whereas in the transverse direction T-tubules are 1.0 μm apart on average (27–29).