Species differences in themorphology of transverse tubule openings in cardiomyocytes

Aims The ultrastructure of ventricular cardiomyocyte T-tubule connections with the outer cell surface (‘mouth’ regions) has been reported to differ between mice and rabbits. In mice, T-tubule mouths form convoluted narrow spaces filled with electron-dense matter that impedes diffusion between T-tubular lumen and bulk extracellular space. Here, we explore whether T-tubule mouths are also constricted in rat (another murine model used frequently for cardiac research) and whether pig and human T-tubule mouth configurations are structurally more similar to mice or rabbits. Methods and results We used chemically-fixed tissue and high-pressure frozen isolated cardiomyocytes to compare T-tubule mouth architecture using transmission electron microscopy and three-dimensional electron tomography. We find that rat T-tubular mouth architecture is more similar to that of rabbits than mice, lacking the marked tortuosity and electron-dense ground substance that obstructs access to deeper portions of the T-tubular system in mice. Pilot observations in larger mammals (pig, human) suggest that mouse may be the least representative animal model of T-tubule connectivity with the outer cell surface in larger mammals. Conclusion Rat T-tubular system architecture appears to be more similar in size and topology to larger mammals than mice. T-tubular mouth topology may contribute to differences in experimental model behaviour, underscoring the challenge of appropriate model selection for research into cell and tissue function

Solute movement in the t-tubule system of rabbit and mouse cardiomyocytes

Cardiac transverse (t-) tubules carry both electrical excitation and solutes toward the cell center but their ability to transport small molecules is unclear. While fluorescence recovery after photobleaching (FRAP) can provide an approach to measure local solute movement, extraction of diffusion coefficients is confounded by cell and illumination beam geometries. In this study, we use measured cellular geometry and detailed computer modeling to derive the apparent diffusion coefficient of a 1-kDa solute inside the t-tubular system of rabbit and mouse ventricular cardiomyocytes. This approach shows that diffusion within individual t-tubules is more rapid than previously reported. T-tubule tortuosity, varicosities, and the presence of longitudinal elements combine to substantially reduce the apparent rate of solute movement. In steady state, large (>4 kDa) solutes did not freely fill the t-tubule lumen of both species and 70 kDa. Detailed model fitting of FRAP data suggests that solute diffusion is additionally restricted at the t-tubular entrance and this effect was larger in mouse than in rabbit. The possible structural basis of this effect was investigated using electron microscopy and tomography. Near the cell surface, mouse t-tubules are more tortuous and filled with an electron-dense ground substance, previously identified as glycocalyx and a polyanionic mesh. Solute movement in the t-tubule network of rabbit and mouse appears to be explained by their different geometric properties, which impacts the use of these species for understanding t-tubule function and the consequences of changes associated with t-tubule disease