ANKYRIN DEPENDENT MITOCHONDRIAL FUNCTION AND BIOENERGETICS IN THE HEART

ANK2 mutations in patients are associated with numerous arrhythmias, cardiomyopathies, and other heart defects. In the heart, AnkB, the protein encoded by ANK2, clusters relevant ion channels and cell adhesion molecules in several important domains; however, its role at Mitochondria Associated ER/SR Membranes (MAMs) has yet to be investigated. MAMs are crucial to mitochondrial function and metabolism and are signaling hubs implicated in various cardiac pathologies. Among several functions, these sites mediate the direct transfer of calcium from the ER/SR to the mitochondria to modulate ATP synthesis. Given that mitochondrial function and energy production are paramount to cardiovascular heath, the work in this thesis explores the role of AnkB in recruiting and tethering wolframin (Wfs-1), a novel ankyrin binding protein along with Inositol Triphosphate Receptor (IP3R), Sigma1-R (Sig1R), and Voltage Dependent Anion Channel (VDAC) at MAMs to constitute a Ca2+ signaling domain. Through a series of cellular fractionation, co-immunoprecipitation, functional assays, and fluorometry, we evaluated AnkB dependent protein complex formation at MAMs and the energetic implications of decreased AnkB in the heart. We found that 3-month-old AnkB+/- mice display lowered cardiac performance along with trends toward cardiac remodeling. Further, we are first to report AnkB expression at MAMs, where it colocalizes with and co-immunoprecipitates Ca2+ regulating proteins including IP3R, Sig1R, Wfs-1, and VDAC. Reduction in AnkB leads to elevated cardiac mitochondrial Ca2+ levels with a host of metabolic implications including increased oxygen consumption through the electron transport chain (ETC), overproduction of reactive oxygen species (ROS), decreased mitochondrial membrane potential (MMP), and inefficient oxidative phosphorylation (OXPHOS)/ATP production. Altogether, these results further elucidate the role of AnkB in the heart and provide novel insights into the mitochondrial aspect of AnkB related cardiac dysfunction

OBSCURIN MEDIATES ANKYRIN COMPLEX FORMATION IN THE HEART

Distinctly organized domains of receptors, ion channels, transporters, signaling molecules, cell adhesion molecules, and contractile proteins are crucial to cardiac function. Interactions between adaptor proteins such as ankyrins and cytoskeletal proteins such as obscurin play a pivotal role in organizing these functional domains in cardiomyocytes. Therefore, dysfunction of both ankyrin as well as obscurin lead to a host of cardiovascular diseases such as arrhythmias and cardiomyopathies. Alternative splicing of ankyrin yields numerous isoforms that interact with obscurin at various sub-cellular domains. And while some of these obscurin-ankyrin complexes have been studied, many others have not been characterized. Further, previous studies have focused on these ankyrin-obscurin complexes individually; however, how ankyrin-mediated macromolecular complexes are integrated together within the cardiomyocyte is not clear. Thus, the work in this thesis describes mechanisms by which obscurin organizes two separate ankyrin-mediated macromolecular complexes simultaneously. Two muscle-specific ankyrin isoforms, sAnk1.5 and AnkG107/130, which do not interact with each other, were used to test if obscurin could serve to bridge these ankyrins. Through a series of in vitro binding assays, co-precipitation assays, and FLIM-FRET analysis we demonstrate that obscurin binds both isoforms simultaneously via interactions with two ankyrin binding sites in its C-terminus. We also show that β1-spectrin, a protein that does not directly interact with obscurin or sAnk1.5, can be recruited to a macromolecular complex via an interaction with AnkG107/130. Lastly, both ankyrin isoforms localize to the M-line of cardiomyocytes suggesting that obscurin could serve to target two separate ankyrin protein complexes to the same domain (M-line). In sum, this work provides a novel, compelling mechanism for ankyrin complex integration and coordination in cardiomyocytes

Study on the Effect of Void Geometry and Location on Electric Field Distribution and Partial Discharges in HVDC Cables

Nowadays HVDC transmission systems are preferred over HVAC systems because of their less power loss and high reliability. In the HVDC cable transmission system, there is a problem of Partial Discharges (PD) occurring due to cavities of the dielectrics in HVDC cables. The distribution of the electric field may change in the presence of partial discharge, which has an impact on how the electric system normally functions. Under DC conditions, partial discharge characteristics are influenced by the electric field distribution and electrical conductivity. Discharges tend to occur quickly when the cavity field reaches the inception level. Hence it is necessary to monitor the conditions of the HVDC cable for better transmission. A 2-D FEM-based model is developed for the simulation of PD due to various shapes of voids at various locations in the cable insulation using ANSYS. Also, PD modeling is the most important concern in identifying the influencing parameters and physical mechanisms of the PD phenomenon. In this work, the Three capacitances model is used to estimate the model parameters and simulate the partial discharges in MATLAB. The results from the simulation are compared with analytical models. It is observed that pulses of the partial discharges vary with the dimensions of the void, type of void materials, and location of the void

Epigenetic regulation of the nuclear genome associated with mitochondrial dysfunction in Leber’s hereditary optic neuropathy (LHON)

Abstract Leber’s hereditary optic neuropathy (LHON) is a mitochondrial hereditary disease in which visual loss affects complex 1 activity of the electron transport chain of mitochondria. It first manifests as painless dulling or blurry in one or even both eyes, and as it develops, sharpness and color perception are lost. In addition to primary mitochondrial DNA (mtDNA) mutations, there are also other environmental and epigenetic factors involved in the pathogenesis of LHON. One of the most common locations for deadly pathogenic mutations in humans is the human complex I accessory NDUFS4 subunit gene. The iron-sulfur clusters of the electron input domain were distorted in the absence of NDUFS4, which reduced complex I function and elevated the production of reactive oxygen species. Therefore, here, we studied the epigenetic alterations of NDUFS4 by focusing on histone activation and repressive markers. We isolated peripheral blood mononuclear cells (PBMCs) from LHON patients and healthy individuals and examined epigenetic modifications in ND4 mutant cells and control cells. Chromatin immunoprecipitation-qRT PCR (ChIP-qRT PCR) assays were performed to investigate the modifications of histones. In comparison to their controls, both LHON patients and ND4 mutant cells exhibited a significant enrichment in activation and repressive markers. This finding indicates that these modifications might mitigate the impact of LHON mutations on complex 1 and aid in elucidating the mechanism underlying the progression of LHON disease