Examination of the Transverse Tubular System in Living Cardiac Rat Myocytes by 2-Photon Microscopy and Digital Image–Processing Techniques

Abstract

The transverse tubular system (t-system) of cardiac muscle is a structure that allows rapid propagation of excitation into the cell interior. Using 2-photon molecular excitation microscopy and digital image-processing methods, we have obtained a comprehensive overview of the t-system of rat ventricular myocytes in living cells. We show that it is possible to quantify the morphology of the t-system in terms of average local tubule diameter, branching pattern, and local abundance of the t-system by immersing living myocytes in a dextran-linked fluorescein solution. Our data suggest that previous electron microscopic examinations of t-system structure have underestimated both the geometric complexity of the t-system morphology and the fraction of cell volume occupied by the t-system (3.6% in this species). About 40% of tubules occur between Z-lines, and the t-tubule diameter is 255+/-0.85 nm (mean+/-SEM). The t-tubules leave the outer surface of the cell in an approximately rectangular array; however, at some points junctions between the t-tubules and the surface membrane are missing. In view of the complexity of the t-system apparent from our images, we propose that the t-system be renamed the "sarcolemmal Z rete." The methods presented here are generally applicable to the quantification of the sarcolemmal Z rete and other structures within cells by fluorescence microscopy in a variety of cell types.

Quantification of t-tubule area and protein distribution in rat cardiac ventricular myocytes

“…Thus spaces in the image were taken to be part of the t-tubular network, an assumption supported by their dimensions and proximity to the Z line ( fig. 2, right), which are similar to those reported previously (Soeller & Cannell, 1999), although it is not possible to determine whether they are open to the extracellular space. The EM images in figure 2 show that the gross structure of the cell appears to be well maintained following detubulation, although there are fewer apparent t-tubules in the formamidetreated cell; table 1 shows that the main change following formamide treatment is a decrease in the number of t-tubules per unit area, although the area of each t-tubule appears to be unchanged.…”

“…This is similar to the fraction of cell membrane estimated to be in the ttubules using electron microscopy (Page, 1978). However structural and sampling distortions can occur during preparation of tissue for electron microscopy, and measurements obtained by filling the t-tubules of a living cell with fluorescent dye (Soeller & Cannell, 1999) combined with measurements of surface/volume ratio (Satoh et al, 1996) suggest that >50% of the cell membrane is within the t-tubules (Bers, 2001). We have used a simple model to explore this discrepancy: we determined the combinations of t-tubule radius, length and density that produce either 32% (the value obtained by detubulation) or 56% (a value computed from optical measurements and the average surface/volume ratio of 0.782 μm 2 /μm 3 from Satoh et al 1996;above).…”

“…The planes in figure 1 show solutions to this equation over the range of ttubule radii and densities reported experimentally. These data show that the plane produced using a fractional area of 56 % contains tubular lengths in the range reported for the average length of tubular inter-branch segments (average 6.9 µm; Soeller & Cannell, 1999), whereas a fractional area of 32% produces lengths <4 µm, which is less than half the diameter of a cardiac myocyte and shorter than observed experimentally. These data suggest that a fractional value of 56% may be closer to the actual value than 32%.…”

“…However optical measurements suggest that the surface area of the t-tubules is 0.44 μm 2 per μm 3 cell volume (Soeller & Cannell, 1999); normalized to the estimated total surface area/cell volume of a myocyte 100 μm long and 20 μm in diameter (0.68 μm 2 /μm 3 ) this gives a t-tubular membrane fraction of 65% (Soeller & Cannell, 1999). This agrees with the lower capacitance (pF)/ cell volume (pl) ratio determined by Satoh et al (1996;6.76 pF/pl in 3 month old rats, compared to 8.88 pF/pl in 6 month old rats): assuming a membrane capacitance of 1 µF/cm 2 these values give surface area/volume ratios of 0.676 and 0.888 µm 2 /µm 3 , and hence t-tubular fractions of 65% and 50%, respectively.…”

On-chip acidification rate measurements from single cardiac cells confined in sub-nanoliter volumes

“…Furthermore, the Na+ /H+ exchanger is localized at the intercalated disks (faces of the brick shaped cardiac myocytes) and the transverse tubules (Petrecca et al, 1999). The tubules are hollow vessels perpendicular to the longitudinal surface and occupy a 64% of the total surface membrane (Soeller and Cannell, 1999) leaving only approximately 2% for the intercalated disk facing quasi-reference electrode. Therefore, the proton flux over the cell surface is inhomogeneous and hypothesized to be dominated by circumference in close proximity of our IrO x sensing electrodes raising the pH locally as confirmed by our measurements.…”