Mechanisms of Osmotically-Induced T-tubule Remodeling in Mouse Cardiomyocytes

Abstract

T-tubules are invaginations of the surface sarcolemma that form a complex, interconnected network and are distinctive structures of adult ventricular cardiomyocytes. T-tubules are intimately linked to the intracellular sarcoplasmic reticulum to form structures called dyads, which house proteins critical for excitation-contraction coupling. Disruption of the t-tubule network, particularly t-tubule loss, causes contractile dysfunction and has been extensively characterized in various cardiovascular diseases. Multiple studies have correlated mechanical stress associated with cardiovascular diseases with t-tubule remodeling but the underlying causal mechanism remains unknown. The overall goal of this thesis is to study the function of t-tubules and the factors that contribute to their remodeling. This thesis tests the central hypothesis that sub-microscopic t-tubular structures (constrictions and dilations) regulate the diffusional properties and stability of t-tubules in response to osmotic stress. Generally, t-tubules have been modeled as cylindrical tubes and although constrictions and dilations within t-tubules have been well documented, the contributions of these sub-microscopic structures to cardiac physiology has been largely unrecognized. A novel fluorescence based assay was developed to characterize the diffusional properties of cardiac t-tubules, which depend on the fine t-tubular structure. Quantitative measurements of dextran diffusion in t-tubules combined with computational modeling revealed that t-tubular constrictions and dilations make significant contributions to the apparent diffusion coefficient of molecules within the t-tubular lumen and to the electrical properties of t-tubules. Subsequent experiments demonstrated that osmotic detubulation is a threshold phenomenon and provided quantitative insights into the mechanism of detubulation. A series of conditions were developed to induce t-tubular constrictions through osmotic stress and chemical modifications. Next, I tested the hypothesis that t-tubule constrictions predispose t-tubules to seal following osmotic stress. A strong correlation between the diffusional accessibility of t-tubules and the magnitude of t-tubule sealing following osmotic stress was established, suggesting that constrictions and dilations strongly modulate the stability of t-tubules. Finally, we found that the concentration gradient of small membrane permeable molecules (SMPMs) across the membrane could strongly affect the susceptibility of t-tubules to detubulation. We proposed that the “bilayer-couple hypothesis”, which describes the formation of membrane curvature due to membrane area asymmetry across the membrane, can effectively explain the modulatory roles of SMPMs. These results support the notion that the sub-microscopic structure of t-tubules play a key role in determining their stability in response to osmotic stresses. Overall, we have identified a number of critical factors that regulate t-tubular stability and determine the response of t-tubules to stress. The results presented in this dissertation demonstrate the functional and structural significance of sub-microscopic t-tubular structures. Furthermore, the contribution of sub-microscopic t-tubular structures must be taken into account when developing future models of t-tubular remodeling.