Three-dimensional distribution of cardiac Na+-Ca2+ exchanger and ryanodine receptor during development

Abstract

Mechanisms of cardiac excitation-contraction coupling in neonates are still not clearly defined. Previous work in neonates shows reverse-mode Na(+)-Ca(2+) exchange to be the primary route of Ca(2+) entry during systole and the neonatal sarcoplasmic reticulum to have similar capability as that of adult in storing and releasing Ca(2+). We investigated Na(+)-Ca(2+) exchanger (NCX) and ryanodine receptor (RyR) distribution in developing ventricular myocytes using immunofluorescence, confocal microscopy, and digital image analysis. In neonates, both NCX and RyR clusters on the surface of the cell displayed a short longitudinal periodicity of approximately 0.7 microm. However, by adulthood, both proteins were also found in the interior. In the adult, clusters of NCX on the surface of the cell retained the approximately 0.7-microm periodicity whereas clusters of RyR adopted a longer longitudinal periodicity of approximately 2.0 microm. This suggests that neonatal myocytes also have a peri-M-line RyR distribution that is absent in adult myocytes. NCX and RyR colocalized voxel density was maximal in neonates and declined significantly with ontogeny. We conclude in newborns, Ca(2+) influx via NCX could potentially activate the dense network of peripheral Ca(2+) stores via peripheral couplings, evoking Ca(2+)-induced Ca(2+) release.

FIGURE 1

NCX distribution in 3d and 10d ventricular myocytes. (A) 3d. (Aa, top) 3-D reconstruction of NCX distribution; (bottom) a cross section of top image; section thickness = 5 μm; scale bar = 5 μm. (Ab) Magnified NCX label at cell surface, scale bar = 2 μm. (Ac) Single optical section, scale bar = 5 μm; (inset) magnified region of surface label. (B) 10d myocyte. (Ba, top) 3-D reconstruction of NCX distribution; (bottom) a cross section of top image; single arrows point to surface invagination; scale bar = 5 μm. (Bb, top) Single optical section (single arrow points to vesicular-like structures); (bottom) a magnified view of vesicular-like structure. (Bc, top) Magnified side view (yz) of a vesicular structure with no link to sarcolemma (single arrow, structure; double arrow, surface membrane; scale bar = 2 μm); (bottom) magnified side view (yz) of a structure with an apparent link to sarcolemmal invagination (single arrow, structure; double arrow, surface membrane invagination; triple arrow, surface membrane).

FIGURE 2

NCX distribution in 20d and 56d ventricular myocytes. (A) 20d myocyte. (Aa, top) 3-D reconstruction of NCX distribution; (bottom) a cross section of top image (single arrow points to surface membrane invaginations), scale bar = 5 μm. (Ab) Single optical section (single arrow, vesicular-like structures; double arrows, surface membrane invaginations; scale bar =5 μm). (B) 56d myocyte. (Ba, top) 3-D reconstruction of NCX distribution; (bottom) a cross section of top image, scale bar = 5 μm. (Bb) Single optical section at cell center (single arrow, closely spaced clusters; double arrows, widely spaced clusters; triple arrows, NCX label between the Z-lines, scale bar = 5 μm. (Bc) A magnified view of surface NCX label; scale bar = 2 μm.

FIGURE 3

RyR distribution in 3d and 10d ventricular myocytes. (A) 3d myocyte. (Aa, top) 3-D reconstruction of RyR distribution; (bottom) a cross section of top image; single arrow, peripheral label; double arrows, internal label; scale bar = 5 μm. (Ab) Magnified RyR label at cell surface, scale bar = 2 μm. (Ac) Single optical section (single arrow, orderly spaced internal RyR; double arrows, sparsely spaced internal RyR; n, nuclear region); (inset) magnified view of surface label. (B) 10d myocyte. (Ba, top) 3-D reconstruction of RyR distribution; (bottom) a cross section of top image; scale bar = 5 μm. (Bb) Single optical section at cell center (single arrow, closely spaced peripheral RyR; double arrows, widely spaced peripheral RyR; n, nuclear region; scale bar = 5 μm).

FIGURE 4

RyR distribution in 20d and 56d ventricular myocytes. (A) 20d myocyte. (Aa, Top) 3-D reconstruction of RyR distribution; (bottom) a cross section of top image; scale bar = 5 μm. (Ab) Single optical section (single arrow, label between the Z-line; double arrows, absence of label between Z-line; scale bar = 5 μm. (B) 56d myocyte. (Ba, top) 3-D reconstruction of RyR distribution; (bottom) a cross section of top image, scale bar = 5 μm. (Bb) Magnified RyR label at cell surface, scale bar = 2 μm. (Bc) Single optical section (single arrow, label between Z-line; double arrows, absence of label between Z-line; scale bar = 5 μm).

FIGURE 5

Cell layering and outline delineation. (A) Cross section of a 3d myocyte labeled for NCX (red) divided into layers (white lines; not drawn to scale) across the cell diameter; scale bar = 5 μm. (B) Single optical plane of a 3d ventricular myocyte stained for NCX (white). A 3-pixel-thick band (red; not drawn to scale) outlines the cell boundary.

FIGURE 6

NCX or RyR fraction at different cell layers. (A) NCX fraction at different layers. (B) RyR fraction at different layers. (C) NCX, (D) RyR, or (E) colocalized voxel, fraction at surface (layers 1, 2, and 3) versus interior (remaining layers) compartment; * denotes a significant difference between surface and interior (p < 0.05); # denotes a significant difference in surface distribution between groups (p < 0.05), except for between 3d and 6d; + denotes a significant difference in interior distribution between groups (p < 0.05), except for between 3d and 6d.

FIGURE 7

Intercluster distance of surface NCX or peripheral RyR. (A) Probability distribution of NCX intercluster distances; y axis denotes the probability of a particular intercluster spacing occurring within a cell; * denotes a significant difference between the 56d and the younger groups (p < 0.05). (B) Probability distribution of RyR intercluster distances well described by the sum of two Gaussian functions. The fits show each age group has two peak probabilities, denoted as left or right peak. (C) Peripheral RyR intercluster distance corresponded to the left or the right peak probability shown in panel B.

FIGURE 8

NCX and RyR colocalization in developing and matured ventricular myocytes. Merged images of NCX (red) and RyR (green) label previously shown; colocalized voxels are white; scale bar = 5 μm.

FIGURE 9

Whole cell NCX and RyR colocalization versus colocalized voxel density in developing and matured ventricular myocytes. (A) Colocalization in whole cell; # denotes a significant difference between 3d versus 10d, 20d, and 56d (p < 0.05); * denotes a significant difference between groups; + denotes a significant difference between groups (p < 0.05). (B) Colocalized voxel density (number of colocalized voxels per pL surface compartment); * denotes a significant difference between 3d versus 10d, 20d, and 56d (p < 0.05).