Three-dimensional reconstruction and analysis of the tubular system of vertebrate skeletal muscle

Abstract

Skeletal muscle fibres are very large and elongated. In response to excitation there must be a rapid and uniform release of Ca2+ throughout for contraction. To ensure a uniform spread of excitation throughout the fibre to all the Ca2+ release sites, the muscle internalized the plasma membrane, to form the tubular (t-) system. Hence the t-system forms a complex and dense network throughout the fibre that is responsible for excitation-contraction coupling and other signalling mechanisms. However we currently do not have a very detailed view of this membrane network due to limitations in previously used imaging techniques to visualize it. Here we serially image fluorescent dye trapped in the t-system of fibres from rat and toad muscle on the confocal microscope, and deconvolve and reconstruct these images to produce the first 3 dimensional reconstructions of large volumes of the vertebrate t-system. Large volume reconstructions showed complex arrangements of tubules that have not been described previously and also allowed the association of the t-system with cellular organelles to be visualized. A high density of tubules close to the nuclear envelope was observed due to the close and parallel alignment of the long axes of the myofibrils and the nuclei. Furthermore local fluorescence intensity variations from sub-resolution tubules were converted to tubule diameters. Mean diameters of tubules were 85.9 ± 6.6 and 91.2 ± 8.2 nm, from rat and toad muscle under isotonic conditions, respectively. Under osmotic stress the distribution of tubular diameters shifted significantly in toad muscle only, with change specifically occurring in the transverse but not longitudinal tubules.