||In this thesis I present novel findings of microstructural remodeling that occurs during dyssynchronous heart failure (DHF) and the ability for cardiac resynchronization therapy (CRT) to reverse this remodeling. DHF is an advanced disease state that occurs in a large portion of patients suffering from heart failure. Mechanical dyssynchrony between the left and right ventricles of the heart, the hallmark of DHF, results in significantly increased heterogeneity of stress in the cardiac wall. DHF severely limits cardiac performance, decreasing quality of life and increasing mortality. The main therapy for treating DHF is CRT, a therapy in which mechanical synchrony is restored to the ventricles via electrical pacing. The success of CRT varies widely. Scientific knowledge surrounding DHF and CRT is surprisingly sparse for how widespread the disease and therapy are. A better understanding of the subcellular structure and function altered during DHF will improve our understanding of the disease and potentially help develop novel therapies and even lead to development of assays capable of better predicting success of current therapies. Here we use confocal microscopy to explore protein distributions within isolated cardiomyocytes and intact tissue, Ca2+ handling during activation and relaxation of stimulated cardiomyocytes, and to develop a method for quantifying strain in 2D image sequences of contracting cardiomyocytes at an unprecedented spatiotemporal resolution. Specifically I will demonstrate that ?-actinin, the protein comprising the majority of the sarcomeric Z-disk, is significantly altered during DHF and that CRT is able to partially reverse this remodeling. I will then present findings on remodeling of the transverse tubular system and associated ryanodine receptor clusters, both crucial components of excitation-contraction coupling. In particular, I will show that these structures exhibit subcellular heterogeneity during DHF, affecting excitation-contraction coupling. This heterogeneity is reduced after CRT, indicating previously unknown capabilities of restoration. Finally, I will present a novel method to characterize strain within contracting cardiomyocytes. This method expands on previous methods by providing a regional 2D strain tensor at unprecedented spatiotemporal resolution, allowing more accurate description of the mechanical properties of the cell. Together, this work makes a significant contribution to the understanding of DHF and CRT.