Towards a cellular architecture of three-dimensional array of muscle fibers

Abstract

This thesis presents a novel method for reconstructing three-dimensional myofiber orientation across entire mouse ventricular walls at the micrometer scale, significantly improving upon the limited spatial resolution of existing methods. This resolution improvement enables a comprehensive understanding of myofiber geometry and reveals a new fiber system, which has remained elusive due to the limitations of existing imaging techniques. The methodology combines tissue clearing (CLARITY), high-resolution confocal microscopy, and advanced computer vision techniques to reconstruct myofibers across entire mouse ventricular walls at the micron scale resolution. From the fluorescence signal at cardiomyocyte boundaries, this method extracts information where the intensity gradient provides unbiased estimates of the eigenvectors associated with the structure tensor. The resulting reconstructions reveal a complex geometry of myofibers, including prominent long-axis fibers that are orthogonal to the well-known circumferential ones. These findings corroborate previous studies that employed lower-resolution methods but provide unprecedented detail about the three-dimensional organization of myofibers. Moreover, our methodology led to the discovery of an additional cell layer in the outer ventricular wall, a significant finding that reshapes our understanding of heart wall structure. This cellular layer lies in a thin shell and forms a continuum with longitudinally arranged cardiomyocytes in the inner walls, with a complex geometry at the apex. The findings from this study pave the way for the investigation of myofiber remodeling in heart diseases, providing a valuable tool for elucidating the mechanisms underlying cardiac dysfunction associated with structural changes. By unraveling the intricate organization of myofibers, this work has significant implications for our understanding of heart function and cardiac diseases.