Abstract 17093: The 3D T-Tubule Network Significantly Remodels in Cardiomyocytes Subjected to Mechanical Load

Abstract

Introduction: T-tubules (TT) are critical for excitation-contraction coupling in cardiomyocytes (CMs). Past work studying heart failure has spatially localized TT loss with inhibited Ca2+ release in the 2D space. This limited view does not consider tubules in other z-planes, necessitating a 3D understanding of the network. Hypothesis: We hypothesize that the ultrastructure of the 3D TT network significantly remodels in response to increase in culture substrate stiffness. Methods: Adult rat ventricular CMs were cultured ex vivo on substrate with either physiological stiffness (10 kPa) or pathological stiffness (50 kPa) for 2 days. Live CMs were stained for TTs (Di-8-ANEPPS) and Ca2+ (Fluo-4). 3D renderings of the TT network were reconstructed from z-stacks of 2D confocal images in the Arivis software. Within a set 3D region of interest, orientation (longitudinal, transverse, axial), volume, and surface area of tubules were measured. Ca2+ transients were taken from a line scan. Data from 18 CMs cultured at 10 kPa and 15 CMs cultured at 50 kPa were acquired. Results: CMs cultured on 50 kPa surfaces exhibited a decrease in the number of transverse elements (147 to 112) and an increase for longitudinal elements (3 to 16) compared to control CMs cultured on 10 kPa surfaces. For all 3 orientations, the volume of the average tubule increased by at least 20% following culture on 50 kPa surfaces. The total surface area of all axial and longitudinal tubules increased by at least 130% and decreased by 13% for all transverse tubules following culture on 50 kPa surfaces. Conclusions: Two days of culture on stiffened surfaces, designed to mimic the stiffness of diseased hearts, produces significant TT remodeling. Our work suggests that the remodeled TT network has less spatial capacity to release Ca2+ and worsened access to actin-myosin sites. In the future, we will associate the distance between each pixel on the line scan to the nearest TT with Ca2+ release parameters. This model system will permit further elucidation of how particular aspects of TT remodeling affect Ca2+ release and will facilitate testing of therapeutic interventions designed to prevent load-dependent defects in excitation-contraction coupling.