Examination of the Transverse Tubular System in Living Cardiac Rat Myocytes by 2-photon Microscopy and Digital Image-processing Techniques

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.